He was baptized at Naughton, Suffolk in 1593. He was a student at Emmanuel College, Cambridge from 1607, graduating Bachelor of Arts in 1611, Master of Arts 1614, Bachelor of Divinity 1621, and Doctor of Divinity 1626. A Fellow from 1616, he became rector of Aller in Somerset in 1624, and then succeeding Thomas Taylor he preached at Street Mary, Aldermanbury in London from 1632.
During the 1630s Stoughton came under suspicion from the authorities, and his mail was watched.
His numerous correspondents included John Forbes, John Winthrop, Stephen Marshall, Samuel Ward and William Sandcroft (his old tutor). In 1635 he was before William Juxon, the Bishop of London for supposed nonconformity, with John Goodwin and Sidrach Simpson.
In 1636 he was caught up in the investigation of John White of Dorchester, that involved also Henry Whitefield. With the support of Sir Robert Harley and other patrons Stoughton managed to avoid serious problems.
At the end of his life Stoughton came into contact with Samuel Hartlib.
His millennial pamphlet Felicitas ultimi saeculi was taken to Hungary in 1638 by John Tolnai, a contact of Comenius. lieutenant was intended for György Rákóczi. Two years later, after Stoughton"s death, Hartlib published the pamphlet with Stoughton"s covering letter.
Doctor Stoughton thereby became step-father to the New England colonist James Cudworth, to Ralph Cudworth the younger (whom he educated in preparation for a place at the University of Cambridge), and to the other Cudworth children.
Mary died in 1634, and Doctor Stoughton then married Jane Browne, widow of Walter Newburgh, and daughter of John Browne of Frampton, Dorset, in 1636.