Born at Crevalcore in Italy, Malpighi graduated firstly in philosophy and then medicine from the University of Bologna. He subsequently served as professor of medicine at Pisa, Bologna, and Messina. In later life he became private physician to Pope Innocent XII.

Malpighi may be considered the father of microscopy: he was one of the first to reveal the hitherto hidden world of the detailed structure of animals and plants. Some of his most important investigations were concerned with mammalian blood. In 1660 he provided a clue to the mechanism of respiration by his discovery that blood flowed over the lungs by means of a complex system of minute vessels. He also discovered the fine capillaries or blood vessels in the wing membrane of bats by means of the microscope, and demonstrated their linkage with minute arteries and veins, in this way amplifying the blood circulation theories of William Harvey and Malpighi's contemporary Olof Rudbeck. He was also able to locate the filtering units of the vertebrate kidney, which became known as Malpighian bodies or corpuscles, and discovered traces of gill-like structures in chick embryos, although the true nature and significance of his discovery was not recognized until later. Using a chick as his model, he was able to describe the formation of the heart from undifferentiated tissue. Studies of the skin resulted in his description of the layer of epidermis (the Malpighian layer) next to the dermis in which active cell division takes place.

Malpighi was also interested in insect physiology, showing that the insect respiratory system was based on the simple diffusion of gases in vessels called tracheae. In the first published treatise to deal exclusively with an invertebrate animal, he described the internal organs and anatomy of the silkworm moth. In the world of plants, Malpighi noted the stomata on the undersides of leaves, appreciated the differences in development between monocotyledons and dicotyledons, and described the annual rings of dicotyledon stems.

In 1669, Malpighi became the first Italian to be named an honorary member of the Royal Society, following which much of his research was published in the Philosophical Transactions of the society.