Driven abroad about 1593, he found a home in "a blind lane at Amsterdam", acting as "porter" to a bookseller, who, on discovering his knowledge of Hebrew, introduced him to other scholars. When part of the London church, of which Francis Johnson (then in prison) was pastor, reassembled in Amsterdam, Ainsworth was chosen as their doctor or teacher. In 1596 he drew up a confession of their faith, reissued in Latin in 1598 and dedicated to the various universities of Europe (including St Andrews, Scotland). Johnson joined his flock in 1597, and in 1604 he and Ainsworth composed An Apology or Defence of such true Christians as are commonly but unjustly called Brownists.
Organizing the church was not easy and dissension was rife. Though often involved in controversy, Ainsworth was not arrogant, but was a steadfast and cultured champion of the principles represented by the early Congregationalists. Amid all the controversy, he steadily pursued his studies. The combination was so unique that some have mistaken him for two different individuals. (Confusion has also been occasioned through his friendly controversy with one John Ainsworth, who left the Anglican for the Roman Catholic church.)
In 1608 Ainsworth answered Richard Bernard's The Separatist Schisme, but his greatest minor work in this field was his reply to John Smyth (commonly called "the Se-Baptist"), entitled Defence of Holy Scripture, Worship and Ministry used in the Christian Churches separated from Antichrist, against the Challenges, Cavils and Contradictions of Mr Smyth (1609).
In 1610 Ainsworth was forced reluctantly to withdraw, with a large part of their church, from Johnson and those who adhered to him. A difference of principle as to the church's right to revise its officers' decisions had been growing between them; Ainsworth taking the more Congregational view. In spirit he remained a man of peace.
His scholarly works include his Annotations -- on Genesis (1616); Exodus (1617); Leviticus (1618); Numbers (1619); Deuteronomy (1619); Psalms (including a metrical version, 1612); and the Song of Solomon (1623). These were collected in folio in 1627. From the outset the Annotations took a commanding place, especially among continental scholars, establishing a scholarly tradition for English nonconformity.
His publication of Psalms, The Book of Psalmes: Englished both in Prose and Metre with Annotations (Amsterdam, 1612), which includes thirty-nine separate monophonic psalm tunes, constituted the Ainsworth Psalter, the only book of music brought to New England in 1620 by the Pilgrim settlers. Although its content was later reworked into the Bay Psalm Book, it had an important influence on the early development of American psalmody.
Ainsworth died in 1622, or early in 1623, for in that year was published his Seasonable Discourse, or a Censure upon a Dialogue of the Anabaptists, in which the editor speaks of him as a departed worthy.