Barlow was the son of Richard Barlow of Long-gill in the parish of Orton, Eden in Westmorland (now Cumbria). He was educated at Appleby grammar school. Aged 16, he entered Queen's College, Oxford, as a servitor, rising to be a tabarder (scholar). He took his BA degree in 1630 and his MA in 1633, when he was elected a fellow of his college. In 1635 he was appointed metaphysical reader to the university, being seen as a master of casuistry, logic, and philosophy. Among his pupils was John Owen.