Wolff was born in Berlin, Germany, on January 18, 1734, the son of a master tailor. He studied at the Collegium Medicochirugicum in Berlin and at the University of Halle, where he received his medical degree in 1759. The work for which he is best known is his dissertation on the early development of organisms, Theoria generationis (Theory of Generation). That thesis was, however, largely ignored or rejected by his own contemporaries.

The popular view of embryonic development during Wolff's lifetime is known as preformism. This doctrine held that living creatures exist, fully developed, within the egg or sperm. Textbooks of the time often contained diagrams of these tiny homunculi. Therefore, embryonic development involved nothing more than the growth of the homunculus to its full size.

In his dissertation, Wolff presented an entirely different view of embryonic development. He suggested that the embryo begins as undifferentiated material that, over time, develops the specific tissues and organs that are present in a mature organism. He used the example of the tip of a growing plant that is originally undifferentiated material but that eventually develops into leaves, stems, flowers, and other specialized plant parts. Because of these ideas, Wolff is today recognized as one of the founders of the modern science of embryology.

During his lifetime, however, Wolff failed to achieve any level of professional recognition in his native Germany, and in 1764, he accepted a position as professor of anatomy and physiology at Russia's St. Petersburg Academy of Sciences. He spent the rest of his life there pursuing his research in biology. He died from a brain hemorrhage on February 22, 1794.