De Graaf was born in Schoonhoven, Netherlands, on July 30, 1641. He studied medicine at the universities of Utrecht, Leiden, Paris, and Angers, earning his degree from the last of these in 1665. He died at the age of thirty-two on August 21, 1673, apparently a victim of the plague. During his lifetime, he held no academic post, working instead as a private physician. He also carried out a vast amount of independent scientific research, much of which earned the praise of his contemporaries and later scientists.

The earliest of his research involved a study of pancreatic juice in 1664. He developed an ingenious technique for extracting the juice from a living dog by inserting a fistula into its pancreas and collecting the juice in a flask. His analysis of the character and function of pancreatic juice was, however, incorrect.

By 1668, de Graaf had turned his attention to another topic, the reproductive system. His first treatise on the male system in 1668 contained no new information, but his analysis of the female system proved an important step in the understanding of reproductive biology. He was the first to identify and describe the ovary, a name earlier proposed by Johannes van Horne (1621-1670) and Jan Swammerdam.

In his research, de Graaf dissected the ovaries of numerous animals. He found a structure that has come to be known as the Graafian follicle. By observing the ovaries before and after fertilization, de Graaf was able to recognize changes in their structure and to see the release of fertilized ova. However, because the ovum itself was not identified until a century and a half later, de Graaf was unable to explain the phenomena he observed.