John Knox was born sometime between 1505 and 1515 in or near Haddington, the county town of East Lothian. His father, william Knox, was a merchant. All that is known of his mother is that her maiden name was Sinclair and that she died when John Knox was a child. Their eldest son, william, carried on his father's Business, which helped in Knox's international communications.

Knox first appears in public records as a priest and a notary in 1540. He was still serving in these capacities as late as 1543 when he described himself as a "minister of the sacred altar in the diocese of St. Andrews, notary by apostolic authority" in a notarial deed dated 27 March. Rather than taking up parochial duties in a parish, he became tutor to two sons of Hugh Douglas of Longniddry. He also taught the son of John Cockburn of Ormiston. Both of these lairds had embraced the new religious ideas of the Reformation.

Knox had avoided being arrested by Lord Bothwell through Wishart's advice to return to tutoring. He took shelter with Douglas in Longniddry. Several months later he was still in charge of the pupils, the sons of Douglas and Cockburn, who wearied of moving from place to place while being pursued. He toyed with the idea of fleeing to Germany and taking his pupils with him. While Knox remained a fugitive, Beaton was murdered on 29 May 1546, within his residence, the Castle of St Andrews, by a gang of five persons in revenge for Wishart's execution. The assassins seized the castle and eventually their families and friends took refuge with them, about a hundred and fifty men in all. Among their friends was Henry Balnaves, a former secretary of state in the government, who negotiated with England for the financial support of the rebels. Douglas and Cockburn suggested to Knox to take their sons to the relative safety of the castle to continue their instruction in reformed doctrine. Knox arrived at the castle on 10 April 1547.

After his return to Geneva, Knox was chosen to be the minister at a new place of worship petitioned from Calvin. In the meantime, Elizabeth Bowes wrote to Knox, asking him to return to Margery in Scotland, which he did at the end of August. Despite initial doubts about the state of the Reformation in Scotland, Knox found the country significantly changed since he was carried off in the galley in 1547. When he toured various parts of Scotland preaching the reformed doctrines and liturgy, he was welcomed by many of the nobility including two Future regents of Scotland, the Earl of Moray and the Earl of Mar.

In summer 1548, the galleys returned to Scotland to scout for English ships. Knox's health was now at its lowest point due to the severity of his confinement. He was ill with a fever and others on the ship were afraid for his life. Even in this state, Knox recalled, his mind remained sharp and he comforted his fellow prisoners with hopes of release. While the ships were lying offshore between St Andrews and Dundee, the spires of the parish church where he preached appeared in view. James Balfour, a fellow prisoner, asked Knox whether he recognised the landmark. He replied that he knew it well, recognising the steeple of the place where he first preached and he declared that he would not die until he had preached there again.

On his release, Knox took refuge in England. The Reformation in England was a less radical movement than its Continental counterparts, but there was a definite breach with Rome. The Archbishop of Canterbury, Thomas Cranmer, and the regent of King Edward VI, the Duke of Somerset, were decidedly Protestant-minded. However, much work remained to bring reformed ideas to the clergy and to the people. On 7 April 1549, Knox was licensed to work in the Church of England. His first commission was in Berwick-upon-Tweed. He was obliged to use the recently released Book of Common Prayer, which maintained the structure of the Sarum Rite while adapting the content to the doctrine of the reformed Church of England. Knox, however, modified its use to accord with the doctrinal emphases of the Continental reformers. In the pulpit he preached Protestant doctrines with great effect as his congregation grew.

Towards the end of 1550, Knox was appointed a preacher of St Nicholas' Church in Newcastle upon Tyne. The following year he was appointed one of the six royal chaplains serving the King. On 16 October 1551, John Dudley, 1st Duke of Northumberland, overthrew the Duke of Somerset to become the new regent of the young King. Knox condemned the coup d'état in a sermon on All Saints Day. When Dudley visited Newcastle and listened to his preaching in June 1552, he had mixed feelings about the fire-brand preacher, but he saw Knox as a potential asset. Knox was asked to come to London to preach before the Court. In his first sermon, he advocated a change for the second edition of the Book of Common Prayer. The liturgy required worshippers to kneel during communion. Knox and the other chaplains considered this to be idolatry. It triggered a debate where Archbishop Cranmer was called upon to defend the practice. The end result was a compromise in which the famous Black Rubric, which declared that no adoration is intended while kneeling, was included in the second edition.

Soon afterwards, Dudley, who saw Knox as a useful political tool, offered him the bishopric of Rochester. Knox refused, and he returned to Newcastle. On 2 February 1553 Cranmer was ordered to appoint Knox as vicar of Allhallows Church in London placing him under the authority of the Bishop of London, Nicholas Ridley. Knox returned to London in order to deliver a sermon before the King and the Court during Lent and he again refused to take the assigned post. Knox was then told to preach in Buckinghamshire and he remained there until Edward's death on 6 July. Edward's successor, Mary Tudor, re-established Roman Catholicism in England and restored the Mass in all the churches. With the country no longer safe for Protestant Preachers, Knox left for the Continent in January 1554 on the advice of friends. On the eve of his FLIGHT, he wrote:

In a letter dated 24 September 1554, Knox received an invitation from a congregation of English exiles in Frankfurt to become one of their ministers. He accepted the call with Calvin's blessing. But no sooner had he arrived than he found himself in a conflict. The first set of refugees to arrive in Frankfurt had subscribed to a reformed liturgy and used a modified version of the Book of Common Prayer. More recently arrived refugees, however, including Edmund Grindal, the Future Archbishop of Canterbury, favoured a stricter application of the book. When Knox and a supporting colleague, william Whittingham, wrote to Calvin for advice, they were told to avoid contention. Knox therefore agreed on a temporary order of Service based on a compromise between the two sides. This delicate balance was disturbed when a new batch of refugees arrived that included Richard Cox, one of the principal authors of the Book of Common Prayer. Cox brought Knox's pamphlet attacking the Emperor to the attention of the Frankfurt authorities, who advised that Knox leave. His departure from Frankfurt on 26 March 1555 marked his final breach with the Church of England.

Shortly after Knox sent the letter to the Queen Regent, he suddenly announced that he felt his duty was to return to Geneva. In the previous year on 1 November 1555, the congregation in Geneva had elected Knox as their minister and he decided to take up the post. He wrote a final letter of advice to his supporters and left Scotland with his wife and mother-in-law. He arrived in Geneva on 13 September 1556.

Though the Queen Regent, Mary of Guise, made no move against Knox, his activities caused concern among the church authorities. The bishops of Scotland viewed him as a threat to their authority and summoned him to appear in Edinburgh on 15 May 1556. He was accompanied to the trial by so many influential persons that the bishops decided to call the hearing off. Knox was now free to preach openly in Edinburgh. william Keith, the Earl Marischal, was impressed and urged Knox to write to the Queen Regent. Knox's unusually respectful letter urged her to support the Reformation and overthrow the church hierarchy. Queen Mary took the letter as a joke and ignored it.

In the summer of 1558, Knox published his best known pamphlet, The first blast of the trumpet against the monstruous regiment of women. In calling the "regimen" or rule of women "monstruous", he meant that it was "unnatural". Knox states that his purpose was to demonstrate "how abominable before God is the Empire or Rule of a wicked woman, yea, of a traiteresse and bastard". The women rulers that Knox had in mind were Queen Mary I of England and Mary of Guise, the Dowager Queen of Scotland and regent on behalf of her daughter, Mary, Queen of Scots. This biblical position was not unusual in Knox’s day; however, even he was aware that the pamphlet was dangerously seditious. He therefore published it anonymously and did not tell Calvin, who denied knowledge of it until a year after its publication, that he had written it. In England, the pamphlet was officially condemned by royal proclamation. The impact of the document was complicated later that year, when Elizabeth Tudor became Queen of England. Although Knox had not targeted Elizabeth, he had deeply offended her, and she never forgave him.

When additional French troops arrived in Leith, Edinburgh's seaport, the Protestants responded by retaking Edinburgh. This time, on 24 October 1559, the Scottish nobility formally deposed Mary of Guise from the regency. Her secretary, william Maitland of Lethington, defected to the Protestant side, bringing his administrative skills. From then on, Maitland took over the political tasks, freeing Knox for the role of religious leader. For the final stage of the revolution, Maitland appealed to Scottish patriotism to fight French domination. Following the Treaty of Berwick, support from England finally arrived and by the end of March, a significant English army joined the Scottish Protestant forces. The sudden death of Mary of Guise in Edinburgh Castle on 10 June 1560 paved the way for an end to hostilities, the signing of the Treaty of Edinburgh, and the withdrawal of French and English troops from Scotland. On 19 July, Knox held a National Thanksgiving Service at St Giles'.

On 1 August, the Scottish Parliament met to settle religious issues. Knox and five other ministers were called upon to draw up a new confession of faith. Within four days, the Scots Confession was presented to Parliament, voted upon, and approved. A week later, the Parliament passed three acts in one day: the first abolished the jurisdiction of the Pope in Scotland, the second condemned all doctrine and practice contrary to the reformed faith, and the third forbade the celebration of Mass in Scotland. Before the dissolution of Parliament, Knox and the other ministers were given the task of organising the newly reformed church or the Kirk. They would work for several months on the Book of Discipline, the document describing the organisation of the new church. During this period, in December 1560, Knox's wife, Margery, died, leaving Knox to care for their two sons, aged three and a half and two years old. John Calvin, who had lost his own wife in 1549, wrote a letter of condolence.

On 19 August 1561, cannons were fired in Leith to announce Queen Mary's arrival in Scotland. When she attended Mass being celebrated in the royal chapel at Holyrood Palace five days later, this prompted a protest in which one of her servants was jostled. The next day she issued a proclamation that there would be no alteration in the current state of religion and that her servants should not be molested or troubled. Many nobles accepted this, but not Knox. The following Sunday, he protested from the pulpit of St Giles'. As a result, just two weeks after her return, Mary summoned Knox. She accused him of inciting a rebellion against her mother and of writing a book against her own authority. Knox answered that as long as her subjects found her rule convenient, he was willing to accept her governance, noting that Paul the Apostle had been willing to live under Nero's rule. Mary noted, however, that he had written against the principle of female rule itself. He responded that she should not to be troubled by what had never harmed her. When Mary asked him whether subjects had a right to resist their ruler, he replied that if monarchs exceeded their lawful limits, they might be resisted, even by force.

On 13 December 1562, Mary sent for Knox again after he gave a sermon denouncing certain celebrations which Knox had interpreted as rejoicing at the expense of the Reformation. She charged that Knox spoke irreverently of the Queen in order to make her appear contemptible to her subjects. After Knox gave an explanation of the sermon, Mary stated that she did not blame Knox for the differences of opinion and asked that in the Future he come to her directly if he heard anything about her that he disliked. Despite her friendly gesture, Knox replied that he would continue to voice his convictions in his sermons and would not wait upon her.

Knox's final encounter with Mary was prompted by an incident at Holyrood. While Mary was absent from Edinburgh on her summer progress in 1563, a crowd forced its way into her private chapel as Mass was being celebrated. During the altercation, the priest's life was threatened. As a result, two of the ringleaders, burgesses of Edinburgh, were scheduled for trial on 24 October 1563. In order to defend these men, Knox sent out letters calling the nobles to convene. Mary obtained one of these letters and asked her advisors if this was not a treasonable act. Stewart and Maitland, wanting to keep good relations with both the Kirk and the Queen, asked Knox to admit he was wrong and to settle the matter quietly. Knox refused and he defended himself in front of Mary and the Privy Council. He argued that he had called a legal, not an illegal, assembly as part of his duties as a minister of the Kirk. After he left, the councillors voted not to charge him with treason.

When the General Assembly convened in June 1564, an argument broke out between Knox and Maitland over the authority of the civil government. Maitland told Knox to refrain from stirring up emotions over Mary's insistence on having mass celebrated and he quoted from Martin Luther and John Calvin about obedience to earthly rulers. Knox retorted that the Bible notes that Israel was punished when it followed an unfaithful king and that the Continental reformers were refuting arguments made by the Anabaptists who rejected all forms of government. The debate revealed his waning influence on political events as the nobility continued to support Mary.

On 29 July 1565 when Mary married Henry Stuart, Lord Darnley, some of the Protestant nobles, including James Stewart, 1st Earl of Moray, rose up in rebellion. Knox revealed his own objection while preaching in the presence of the new King Consort on 19 August 1565. He made passing allusions on ungodly rulers which caused Darnley to walk out. Knox was summoned and prohibited from preaching while the court was in Edinburgh.

On 9 March 1566, Mary's secretary, David Rizzio, was murdered by conspirators loyal to Darnley. Mary escaped from Edinburgh to Dunbar and by 18 March returned with a formidable force. Knox fled to Kyle in Ayrshire, where he completed the major part of his magnum opus, History of the Reformation in Scotland. When he returned to Edinburgh, he found the Protestant nobles divided over what to do with Mary. Lord Darnley had been murdered and the Queen almost immediately married the chief suspect, the Earl of Bothwell. The indictment of murder thus upon her, she had been forced to abdicate and was imprisoned in Loch Leven Castle. Lord Moray had become the regent of King James VI. Other old friends of Knox's, Lord Argyll and william Kirkcaldy, stood by Mary. On 29 July 1567, Knox preached James VI's coronation sermon at the church in Stirling. During this period Knox thundered against her in his sermons, even to the point of calling for her death. However, Mary's life was spared, and she escaped on 2 May 1568.

The fighting in Scotland continued as a civil war. Lord Moray was assassinated on 23 January 1570. The regent who succeeded him, the Earl of Lennox, was also a victim of violence. On 30 April 1571, the controller of Edinburgh Castle, Kirkcaldy of Grange, ordered all enemies of the Queen to leave the city. But for Knox, his former friend and fellow galley-slave, he made an exception. If Knox did not leave, he could stay in Edinburgh, but only if he remained captive in the castle. Knox chose to leave, and on 5 May he left for St Andrews. He continued to preach, spoke to students, and worked on his History. At the end of July 1572, after a truce was called, he returned to Edinburgh. Although by this time exceedingly feeble and his voice faint, he continued to preach at St Giles'.

After inducting his successor, Lawson of Aberdeen, as minister of St Giles' on 9 November, Knox returned to his home for the last time. With his friends and some of the greatest Scottish nobles around him, he asked for the Bible to be read aloud. On his last day, 24 November 1572, his young wife read from Paul's first letter to the Corinthians. A testimony to Knox was pronounced at his grave in the churchyard of St Giles' by James Douglas, 4th Earl of Morton and newly elected regent of Scotland: "Here lies one who never feared any flesh".

Knox was survived by his five children and his second wife. Nathaniel and Eleazar, his two sons by his first wife, attended St John's College, Cambridge. Nathaniel became a Fellow of St John's but died early in 1580. Eleazar was ordained into the Church of England and served in the parish of Clacton Magna. He also died young, and was buried in the chapel of St John's College in 1591. Knox's second wife, Margaret Stewart, got remarried to Andrew Ker, one of those involved in the murder of David Rizzio. Knox's three daughters also married: Martha to Alexander Fairlie; Margaret to Zachary Pont, son of Robert Pont and brother of Timothy Pont; and Elizabeth to John Welsh, a minister of the Kirk.

Knox was notable not so much for the overthrow of Roman Catholicism in Scotland, but for assuring the replacement of the established Christian religion with Presbyterianism rather than Anglicanism. It was thanks to Knox that the Presbyterian polity was established, though it took 120 years following his death for this to be achieved in 1689. Meanwhile, he accepted the status quo and was happy to see his friends appointed bishops and archbishops, even preaching at the inauguration of the Protestant Archbishop of St Andrews John Douglas in 1571. In that regard, Knox is considered the notional founder of the Presbyterian denomination, whose members number millions worldwide.

In England, Knox met his wife, Margery Bowes (died c. 1560). Her father, Richard Bowes (died 1558), was a descendant of an old Durham family and her mother, Elizabeth Aske, was an heiress of a Yorkshire family, the Askes of Richmondshire. Elizabeth Bowes presumably met Knox when he was employed in Berwick. Several letters reveal a close friendship between them. It is not recorded when Knox married Margery Bowes. Knox attempted to obtain the consent of the Bowes family, but her father and her brother Robert Bowes were opposed to the marriage.

Knox led a busy life in Geneva. He preached three sermons a week, each lasting well over two hours. The services used a liturgy that was derived by Knox and other ministers from Calvin's Formes des Prières Ecclésiastiques. The church in which he preached, the Église de Notre Dame la Neuve—now known as the Auditoire de Calvin—had been granted by the municipal authorities, at Calvin's request, for the use of the English and Italian congregations. Knox's two sons, Nathaniel and Eleazar, were born in Geneva, with Whittingham and Myles Coverdale their respective godfathers.