Isaac Ambrose was born in 1604, the son of Richard Ambrose, vicar of Ormskirk, Lancashire. Entering Brasenose College, Oxford, in 1621, he graduated with a Bachelor of Arts degree in 1624, and was ordained to the ministry. He became vicar of the parish church in Castleton, Derbyshire, in 1627, then served at Clapham, Yorkshire, from 1629 to 1631. The following year he received a Master of Arts degree from Cambridge.
Through the influence of William Russell, Earl of Bedford, Ambrose was appointed one of the king’s four itinerant preachers for Lancashire, and took up residence in Garstang, a Lancashire town between Preston and Lancaster. The king’s preachers were commissioned to preach the Reformation doctrines in an area that was strongly entrenched in Roman Catholicism. Shortly thereafter, he was married