Ray, a blacksmith's son from Black Notley, Essex, attended Braintree Grammar School, where he benefited from a trust established to finance needy scholars at Cambridge University. He graduated in 1648 and became a fellow the following year, but his university career ended with the Restoration: as a Puritan, he refused to take the oath required by the Act of Uniformity and he lost his fellowship in 1662.

His activities as a naturalist were funded thereafter by friends from Cambridge, in particular by Francis Willughby, who helped him with the ambitious project of describing all known living things. From 1663 to 1666 Ray and Willughby traveled through Europe, widening their knowledge of the flora and fauna. On their return Ray moved into Willughby's house so that they could collaborate in writing up the work. In 1667 Ray published a catalog of British plants. In the same year he was elected a fellow of the Royal Society.

Willughby died in 1672 but left money in his will to enable Ray to continue their project. Between 1686 and 1704 Ray published Historia plantarum (History of Plants), a three-volume encyclopedia of plants describing 18,600 species. In it he emphasized the importance in classification of distinguishing between the monocotyledons and the dicotyledons but more importantly he fixed the species as the basic unit in the taxonomic hierarchy.

Ray also attempted to classify the animal kingdom. In 1693 he published a system based on a number of structural characters, including internal anatomy, which provided a more natural classification than those being produced by his contemporaries.

Ray is also remembered for his theological writings, in which he used the homologies he had perceived in nature as evidence for the necessity of an omniscient creator.