John Desborough, the son of a large landowner, was born in Cambridgeshire, in 1608. He trained as an attorney before marrying Jane Cromwell, the sister of Oliver Cromwell.

In 1642 Cromwell appointed Desborough as a quarter-master to his troop of cavalry. In the Civil War he commanded the cavalry at the storming of Bristol and at the battle of Langport in 1645. As a major-general, he also took part in the parliamentary victory at Worcester.

Desborough was elected to the House of Commons in 1654 and was appointed as a member of the Council of State of the Protectorate. The following year he was one of the major-generals given the task of imposing Puritan morality. In this role Desborough was given control of west county.

In 1659 Desborough joined Charles Fleetwood and John Lambert to help remove Richard Cromwell as Lord Protector of the Commonwealth.

On the Restoration Desborough escaped to Holland. He returned to England in 1666 and lived in retirement in Hackney until his death in 1680.