He was the elder son of John Johnson, mayor of Richmond, North Riding of Yorkshire, born at Richmond and baptised there on 27 March 1562. George Johnson was his brother. He matriculated at Christ's College, Cambridge, graduated B.A. 1581, M.A. 1585, and was elected fellow before Lady day 1584.
He was a popular preacher in the university, and a follower of the independent presbyterianism advocated by Thomas Cartwright. On 6 January 1589 he expounded this view in a sermon at Great St. Mary's, Cambridge, claiming that church government by elders is jure divino. With Cuthbert Bainbrigg, also a fellow of Christ's, accused of factious preaching, he on 23 January came up before Thomas Nevile, the vice-chancellor.
Refusing to answer on oath to the articles of accusation, Johnson and Bainbrigg were committed to prison. Johnson gave in written answers which set out his views, but again on 13 March and 18 April declined the oath. Bail was offered by Sir Henry Knevett and Sir William Bowes, but was rejected by the authorities. On 22 May Johnson and Bainbrigg addressed a letter to Lord Burghley, the chancellor; but the vice-chancellor laid the case before the court of high commission, which directed the vice-chancellor and heads to proceed at discretion. A form of recantation was given to Johnson on 19 October and he was required to read it in the pulpit of Great St. Mary's. He made an unconvincing retractation and on 30 October he was expelled from the university. He claimed a right of appeal, and refusing to leave; he was in December again in custody and vainly petitioning Burghley, backed by fellows of colleges.
Johnson went to Middelburg in Zealand, where he became preacher to the English merchants in the Gasthuis Kerk, with a stipend of £200. In 1591 'A Plaine Refutation' of the claims of the establishment, by Henry Barrow and John Greenwood came Johnson’s way. It was an answer to George Gifford, and had been sent privately in 1591 to Middelburg to be printed. The edition was burned by Johnson on the magistrate's authority for suppressing it.
In 1592 Johnson came to London to confer with Barrow and Greenwood, who were then in the Fleet Prison. Greenwood was shortly afterwards transferred to the house of Roger Rippon, and formed, in conjunction with Johnson, a separatist church, independent of other churches, but presbyterian in its internal order, At a meeting in the house of Fox, in Nicholas Lane, Lombard Street, Johnson was chosen pastor, marking his change from stringent reformer to separatist.
Discipline was practised, and the sacraments administered. This conventicle being discovered, Johnson was committed for a time to the Wood Street Counter. To avoid detection the place of assembly was constantly changed. On 5 December 1592 Johnson and Greenwood were arrested in the house of Edward Boyes, a haberdasher on Ludgate Hill; Johnson was imprisoned and was twice examined.
He was a third time arrested at Islington (on Sunday, 4 March 1593) with his father and brother George, and with John Penry. Penry was executed on 29 May. Johnson was detained in the Clink prison, Southwark. While in prison he married in 1594 Thomasine, widow of Boyes, who brought him £300. Attempts made by puritan churchmen through Henry Jacob failed to win him back to the national church.
In 1597 the merchants Abraham and Stephen Van Hardwick, and Charles Leigh of Addington Surrey and London, projected a settlement in the island of Ramea, off Newfoundland. They successfully petitioned for leave to transport four from the separatist church there; the four chosen were Johnson and his brother George, with Daniel Studley and John Clarke. Johnson left Gravesend in the Hopewell on 8 April, with Studley, the other two sailing in the Chancewell. After the expedition, during which the Chancewell was wrecked, Leigh brought them back to England. In the end the group made their way to Amsterdam.
Johnson resumed his pastorate among the exiled separatists, with Henry Ainsworth as doctor (teacher). In 1598 he was concerned in a Latin version (for transmission to continental and Scottish universities) of a confession of faith, drawn up by Ainsworth (1596), and repudiating the common name of Brownist for separatists. Dissensions arose in the community. Thomasine's taste in dress scandalised a section of the church, and Johnson's brother was among them. Ainsworth tried to prevent a breach, but ultimately those who objected were excommunicated as slanderers. Between 1604 and 1606 John Smyth, who had been a member of the London separatist church, came to Amsterdam, bringing a contingent from Gainsborough, Lincolnshire. Smyth soon developed individual views both of church government and public worship, and after 1607 seceded with his adherents. Johnson's Amsterdam church at this point had its own meeting-house and three hundred communicants.
More serious differences arose in 1609 out of the differing views of Johnson and Ainsworth as to the function of the eldership. Johnson made the eldership the seat of authority; Ainsworth vested all authority in the congregation itself, of which the elders were an executive. After much discussion Johnson proposed that the 'congregationalists' should move to Leyden, joining the exile church there (a group that included at some points Robert Parker, Henry Jacob, William Ames and John Robinson). But the compromise fell through, and Ainsworth with his congregation obtained a place for worship two doors away from the meeting-house, and moved there in December 1610. The 'Ainsworthian Brownists' as they were popularly termed, were excommunicated by the 'Franciscan Brownists.' Ainsworth began a lawsuit for the recovery of the meeting-house.
Johnson and his presbyterians moved on to Emden in East Friesland, at some stage; how long the Emden settlement lasted is unknown. Johnson died at Amsterdam, and was buried there on 10 January 1618.
He wrote generally for sale in London. He published:Confessio Fidei Anglorum Quorundam in Belgio, &c., 1598 (anon.); 1607, with additions by Ainsworth.
Answer to Maister H. Jacob his Defence of the Churches and Ministry of England, &c., 1600; appended is An Answer to ... his Treatise concerning the Priestes of the Church of England, &c., 1600.
An Apologie or Defence of svch Trve Christians as are . . . called Brovvnists, &c., 1604 (translated into Dutch, 1612).
An Inquirie and Answer of Thomas White, his Discouery of Brownism, &c., 1605.
Certavne Reasons . . . prouing that it is not lawfull to . . . haue any Spiritual communion with the present Ministerie of the Church of England, &c., 1608 (answered by William Bradshaw, in The Vnreasonablenesse of the Separation, &c., Dort, 1614).
A Brief Treatise containing . . . reasons against Two Errors of the Anabaptists, &c., , reprinted 1645.
A Short Treatise concerning the Exposition of . . "Tell the Church," &c.. 1611.
A Christian Plea, conteyning three Treatises . . . touching the Anabaptists . . . Remonstrants. . .the Reformed Churches, &c., 1617.
He contributed a running commentary to A Treatise on the Ministry (1595) by Arthur Hildersam.