Spallanzani was born at Scandiano in Italy and educated at the Jesuit College, Reggio, before leaving to study jurisprudence at Bologna University. While at Bologna he developed an interest in natural history, which was probably encouraged by his cousin, Laura Bassi, who was professor of physics there. After receiving his doctorate he took minor orders and a few years later became a priest, although he continued to pursue his researches into natural history.

Spallanzani's most important experiments, published in 1767, questioned John Needham's ‘proof’ 20 years earlier of the spontaneous generation of microorganisms. He took solutions in which microorganisms normally breed and boiled them for 30 to 45 minutes before placing them in sealed flasks. No microorganisms developed, demonstrating that Needham's broth had not been boiled for long enough to sterilize it. Opponents of Spallanzani asserted, however, that he had destroyed a vital principle in the air by prolonged boiling. While conducting these experiments, Spallanzani showed that some organisms can survive for long periods in a vacuum: this was the first practical demonstration of anaerobiosis (the ability to live and grow without free oxygen).

In 1768 he submitted papers to the Royal Society on his findings concerning the regeneration of amputated parts in lower animals, and on the strength of this was elected a fellow of the Royal Society. In the same year Maria Theresa of Austria appointed Spallanzani to the chair of natural history at Pavia, which at that time was under Austrian dominion, and here he remained until his death. He was also in charge of the museum at Pavia and made many journeys around the Mediterranean collecting natural-history specimens for the museum.

Spallanzani's research interests covered a wide area and during his career he made important contributions to the understanding of digestion, reproduction, respiration, and blood circulation, as well as sensory perception in bats. He also (in 1785) managed to accomplish the artificial insemination of a dog.