He established himself as the head of the laboratory of the Hospital of Ravenna and became a professor of physics at the local college.

In 1840, by recommendation of François Arago (1786–1853), his teacher at the École Polytechnique, to the Grand-Duke of Tuscany, Matteucci accepted a post of professor of physics at the University of Pisa.

Instigated by the work of Luigi Galvani (1737–1798) on bioelectricity, Matteucci began in 1830 a series of experiments which he pursued until his death in 1865.

Using a sensitive galvanometer of Leopoldo Nobili, he was able to prove that injured excitable biological tissues generated direct electrical currents, and that they could be summed up by adding elements in series, like in Alessandro Volta’s (1745-1827) electric pile.

His work in bioelectricity influenced directly the research developed by Emil du Bois-Reymond (1818–1896), a student of the great German biologist Johannes Peter Müller (1801–1858) in Berlin, who tried the duplicate Matteucci's experiments and ended up discovering the nerve's action potential.