Sir Francis was elder son of Robert Knollys, Usher of the Royal Chamber, and his wife, Lettice daughter of Sir Thomas Peniston. The family is said to have been descended from Sir Robert Knollys the famous soldier (d.1407), but the proofs are wanting and the pedigree cannot be authentically traced beyond Sir Thomas Knollys, Lord Mayor of London in 1399 and 1410, from whom Sir Francis was fifth in descent.

Born about 1511, a few years before his father was given the manor of Rotherfield Greys in Oxfordshire for an annual rental of a single red rose, Francis was largely raised under the auspices of his mother, since his father died when he was only seven. Katherine Knollys soon remarried to Sir Robert Lee of Burston in Buckinhamshire, producing a number of half-siblings. Sir Robert died in 1537 and she then became the second wife of Sir Thomas Tresham of Rushton in Northamptonshire. Francis appears to have received some education at Oxford, but an old assertion that he was for a time a member of Magdalen College is unconfirmed.

Henry VIII extended to Francis Knollys the favour that he had shown to his father and, in 1538, secured for him his the estate of Rotherfield Greys. Acts of Parliament in 1541 and in 1546 attested to this grant, in the second act making his wife joint-tenant with him. At the same time, Francis became one of the gentlemen-pensioners at court and, in 1539, attended Anne of Cleves on her arrival in England. In 1542, he entered the House of Commons for the first time as member for Horsham. At the beginning of Edward VI's reign, he accompanied the English army to Scotland and was knighted by the Commander-in-Chief, the Duke of Somerset, at the camp at Roxburgh on 28th September 1547. Knollys' strong protestant convictions recommended him to the young king and to his sister, the Princess Elizabeth; and he spent much time at court, taking a prominent part, not only in tournaments there, but also in religious discussion. On 25th November 1551, he was present at Sir William Cecil's house, at a conference between Catholics and Protestants respecting the corporeal presence in the Sacrament. About the same date, he was granted the manors of Caversham in Oxfordshire and Cholsey in Berkshire, and Jocelyn Palmer - subsequently a Protestant martyr - entered his household in nearby Reading as a tutor to his children. At the end of 1552, Knollys visited Ireland on public business.

The accession of Queen Mary darkened Knollys' prospects. His religious opinions placed him in opposition to the government and he deemed it prudent to cross to Germany. On his departure, the Princess Elizabeth wrote to his wife - the lady's cousin, Catherine Carey - a sympathetic note, expressing a wish that they would soon be able to return in safety. Knollys first took up his residence in Frankfort, where he was admitted as a church-member on 21st December 1557; but, afterwards, he removed himself to Strasburg. According to Fuller, he "bountifully communicated to the necessities" of his fellow-exiles in Germany and, at Strasburg, he seems to have been on intimate terms with Jewel and Peter Martyr. Before Mary's death, he returned to England and, "as a man of assured understanding and truth, and well affected to the protestant religion," he was admitted to the new Queen Elizabeth's privy council in December 1558. He was soon afterwards made Vice-Chamberlain of the Household and Captain of the Halberdiers. While his wife and her sister became women of the Queen's Privy Chamber. In 1560, Knollys' wife and son, Robert, were granted, for their lives, the manor of Taunton, part of the property of the See of Winchester. In 1559, Knollys was chosen MP for Arundel and, in 1562, for Oxford, of which town he was also appointed Chief Steward. In 1572, he was elected the Member of Parliament for Oxfordshire and sat for that constituency until his death. Throughout his parliamentary career, he was a frequent spokesman for the Government on questions of general politics but, as a zealous puritan, in ecclesiastical matters, he preserved an independent attitude.

Knollys' friendship with the Queen and Cecil led to his employment in many offices of anxious responsibility. In 1563, he was Governor of Portsmouth and was much harassed, in the August, by the difficulties in supplying the needs, in men and money, of the Earl of Warwick who was engaged on his disastrous expedition to Le Havre. In April 1566, Sir Francis was sent to Ireland to control the expenditure of Sir Henry Sidney, the Lord Deputy, who was trying to repress the rebellion of Shane O'Neil, and was much hampered by the interference of court factions at home; but, contrary to Elizabeth's wishes, Knollys found himself compelled to approve Sidney's plans. It was, he explained, out of the question to conduct the campaign against the Irish rebels on strictly economical lines. In August 1564, he accompanied the Queen to Cambridge and was created an MA. Two years later, he went to Oxford, also with his sovereign, and received a like distinction there. In the same year (1566), he was appointed Treasurer of the Queen's Chamber.

In May 1568, Mary, Queen of Scots, fled to England and flung herself on Queen Elizabeth's protection. She had found refuge in Carlisle Castle and the delicate duty of taking charge of the fugitive was entrusted jointly to Knollys and to Henry Scrope, 9th Baron Scrope. On 28th May, Knollys arrived at the castle and was admitted to Mary's presence. At his first interview he was conscious of Mary's powerful fascination. But to her requests for an interview with Elizabeth and for help to regain her throne, he returned the evasive answers which Elizabeth's advisers had suggested to him; and he frankly drew her attention to the suspicions which suggested her involvement in her husband Darnley's murder. A month passed and no decision was reached in London respecting Mary's future. On 13th July, Knollys contrived to remove her, despite "her tragical demonstrations," to Bolton Castle in the Yorkshire Dales, the seat of Lord Scrope. Here he tried to amuse her by teaching her to write and speak English. Knollys' position grew more and more distasteful and, writing on 16th July to Cecil whom he kept well informed of Mary's conversations and conduct, he angrily demanded his recall. But while lamenting his occupation, Knollys conscientiously endeavoured to convert his prisoner to his puritanic views and she read the English prayer-hook under his guidance. In his discussions with her, Sir Francis commended so unreservedly the doctrines and forms of Geneva that Queen Elizabeth, upon learning his line of argument, sent him a sharp reprimand. Writing to Cecil in self-defence (8th August 1568), Knollys described how contentedly Mary accepted his plain speaking on religious topics. In fact, Mary made every effort to maintain good relations with him. Late in August, she gave Sir Francis a present for his wife, desired his wife's acquaintance and wrote to him a very friendly note, her first attempt at English composition. In October, when schemes for marrying Mary to an English nobleman were under consideration, Knollys proposed that his wife's nephew, George Carey, might prove a suitable match. In November the inquiry into Mary's misdeeds, which had begun at York, was reopened at Westminster and Knollys pointed out that he needed a larger company of retainers in order to keep his prisoner safe from a possible attempt at rescue. In December, he was directed by Elizabeth to induce Mary to assent to her abdication of the Scottish throne. In January 1569, he plainly told the English queen that, in declining to allow Mary either to be condemned or to be acquitted on the charges brought against her, she was inviting perils which were likely to overwhelm her and entreated her to leave the decision of Mary's fate to her well-tried councillors. On 20th January, orders arrived at Bolton to transfer Mary to Tutbury, where the Earl of Shrewsbury was to take charge of her. The Scottish queen protested against her the removal in a pathetic note (25th January) to Knollys and intended for Elizabeth's eyes; but, next day, she was forced to leave Bolton and Knollys remained with her at Tutbury until 3rd February. His wife's death then called him home. Mary blamed Elizabeth for the fatal termination of Lady Knollys' illness, attributing it to her husband's extended absence in the north.

In April 1571, Knollys strongly supported the retrospective clauses of the bill for the better protection of Queen Elizabeth, by which any person who had previously put forward a claim to the throne was adjudged guilty of high treason. Next year, he was appointed Treasurer of the Royal Household (13th July) and he entertained Elizabeth at 'Abbey House,' the Royal Palace at the dissolved Reading Abbey, where he often resided by permission of the Crown. The office of treasurer, he retained till his death.

Although Knollys was invariably on good terms, personally, with his sovereign, he never concealed his distrust of her statesmanship. Her unwillingness to take "safe counsel," her apparent readiness to encourage parasites and flatterers, whom he called "King Richard the Second's men," was, he boldly pointed out, responsible for most of her dangers and difficulties. In July 1578, he repeated his warning in a long letter and begged her to adopt straightforward measures so as to avert such disasters as the conquest of the Low Countries by Spain, the revolt of Scotland to France and Mary Stuart, and the growth of Papists in England. He did not oppose the first proposals for the Queen's marriage with Alençon which were made in 1579, but during the negotiations, he showed reluctance to accept the scheme and Elizabeth threatened that "his zeal for religion would cost him dear."

In December 1581, he attended the Jesuit Campion's execution and asked him, on the scaffold, whether he renounced the Pope. He was a commissioner for the trials of Parry the Jesuit in 1585, of Babington and his fellow-conspirators, whom he tried to argue into Protestantism, in 1580, and of Queen Mary at Fotheringay in the same year. He urged Mary's immediate execution in 1587, both in Parliament and in the Council. In April 1589, he was a commissioner for the trial Philip Howard, Earl of Arundel. On 16th December 1584, he introduced into the House of Commons the bill legalising a national association to protect the Queen from assassination. In 1585, he offered to contribute £100, for several years, towards the expenses of the War for the Defence of the Low Countries and renewed the offer, which was not accepted, in July 1586. In 1589, he was placed in command of the land forces of Hertfordshire and Cambridgeshire which had been called together to resist the Spanish Armada. Knollys was interested in the voyages of Frobisher am Drake and took shares in the first and second Cathay expeditions.

Knollys never wavered in his consistent championship of the Puritans. In May 1574, he joined Bishop Grindal, Sir Walter Mildmay and Sir Thomas Smith in a letter to Parkhurst, Bishop of Norwich, arguing in favour of the religious exercises known a 'prophesyings.' But he was zealous in opposition to heresy and, in September 1581, he begged Burghley and Leicester to repress such "anabaptistical sectaries" as member of the '"Family of Love...who do serve the turn of the Papists".

Writing to Whitgift, Archbishop of Canterbury, on 20th June 1584, he hotly condemned the Archbishop's attempts to prosecute puritan preachers in the Court of High Commission as unjustly despotic and treading "the highway to the Pope". He supported Cartwright with equal vehemence. On 24th May 1584, he sent to Burghley a bitter attack on "the undermining ambition and covetousness of some of our bishops," and on their persecutions of the Puritans. Repeating his views, in July 1586, he urged the banishment of all recusants and the exclusion from public offices of all who married recusants. In 1588, he charged Whitgift with endangering the Queen's safety by his Popish tyranny and embodied his accusation in a series of articles which Whitgift characterized as a fond and scandalous syllogism. In the parliament of 1589, he vainly endeavoured to pass a bill against non-residence of the clergy and pluralities. In the course of the discussion, he denounced the claims of the bishops "to keep courts in their own name" and denied them any "worldly pre-eminence." This speech, "related by himself" to Burghley, was published in 1608, together with a letter to Knollys from his friend, the puritan Dr. Reynolds 'or Rainolds,' in which Bishop Bancroft's sermon at St. Paul's Cross (9th February 1589) was keenly criticised. The volume was entitled 'Informations, or a Protestation and a Treatise from Scotland...all suggesting the Usurpation of Papal Bishops.' Knollys' contribution reappeared as 'Speeches used in the Parliament by Sir Francis Knoles' in William Stoughton's 'Assertion for True and Christian Church Policie' (London, 1642). Throughout 1589 and 1590, Sir Francis was seeking, in correspondence with Burghley, to convince the latter of the impolicy of adopting Whitgift's theory of the divine right of bishops. On 9th January 1591, he told his correspondent that he marvelled "how her Majestie can be persuaded that she is in as much danger of such as are called Purytanes as she is of the Papysts". Finally, on 14th May 1591, he declared that he would prefer to retire from politics and political office rather than cease to express his hostility to the bishops' claims with full freedom. In 1593, he was made a Knight of the Garter.

Knollys's domestic affairs, at times, caused him anxiety. In spite of his friendly relations with the Earl of Leicester, he did not approve the Royal favourite's intrigues with his daughter, Lettice, widow of Walter Devereux, the 1st Earl of Essex, and he finally insisted on their marriage at Wanstead on 21st September 1578. The wayward temper of his grandson, Robert Devereux, 2nd Earl of Essex (son of the same daughter by her first husband), was a source of trouble to him in his later years and the Queen seemed inclined to make him responsible for the youth's vagaries. Knollys was created KG in 1593 and died on 19th July 1596. In his will, he is described as 'of Caversham', so it seems likely that the mansion he was building there was either complete or nearing completion at this date. He was buried at Rotherfield Greys and an elaborate monument, with effigies of seven sons, six daughters and his son William's wife, is still standing in the church there. A poem on his death was penned by Thomas Churchyard under the title, 'A Sad and Solemne Funerall' (London, 1596). Two portraits of Knollys and one of his wife are said to have been in possession of a descendant at Fernhill House in Winkfield, in 1776.

Knollys married Catherine, the daughter of William Carey, Esquire of the Body to King Henry VIII, by Mary, the daughter of Sir Thomas Boleyn, Earl of Wiltshire, and sister of Queen Ann Boleyn. Lady Knollys was thus first cousin to Queen Elizabeth and sister to Henry Carey, Lord Hunsdon. She died, aged 39, at Hampton Court Palace, while in attendance on the Queen, on 15th January 1569 and was buried at Royal expense, in April, in St. Edmund's Chapel in Westminster Abbey. Elizabeth keenly felt her loss. A broadside epitaph by Thomas Newton, dated in 1569, belonged to Heber. She left seven sons and five daughters. Of the latter, Letitia (1540-1634) was wife successively of Walter Devereux, Earl of Essex, of Robert Dudley, Earl of Leicester and of Sir Christopher Blount; Cecilia, Maid of Honour to Queen Elizabeth; Elizabeth, married Sir Thomas Leighton, Governor of Guernsey; Ann, married to Thomas, Lord de la Warr; and Catherine, married, firstly, to Gerald FitzGerald, Lord Offaly, and, secondly, Sir Philip Boteler of Watton Woodhall. According to their monument, the couple had two further daughters who presumably predeceased them.

All Knollys' sons were prominent courtiers in his lifetime. They were, according to Naunton, at continual feud with the Norreys family and, aided by Leicester's influence, kept their rivals in subjection until the Earl's death. Henry, the eldest son, obtained the reputation of being a very cultivated and religious - particularly anti-Spanish - privateer and soldier. William, the second son and eventual heir, was created the Earl of Banbury in 1626. Edward, the third son, was elected MP for Oxford on 2nd April 1571 and died about 1580. Then there came Robert, Richard and Francis Junior. Thomas, the seventh son, distinguished himself in the warfare in the Low Countries, acting as Governor of Ostend in 1586 and prominently aiding Peregrine Bertie in the Siege of Bergen in 1588. He married Odelia, daughter of John de Morada, Marquis of Bergen.