De Stefani was born in Padua where his father, Luigi, was a lawyer and nationalist married to Caterina Rigon.
He published his degree thesis on economics, examining the value of work, after which he was appointed to the University of Siena to teach statistics and political economy.
In the meantime, he also started looking at the Apuan Alps, publishing a geological map of southern Calabria.
De Stefani clashed with the geologists Bernardino Lotti and Domenico Zaccagna, with animated sessions at the Tuscan Society of Natural Sciences between 1879 and 1882 which resulted in De Stefani distancing himself from Meneghini and his resignation from the Society.
Among his ideas was that the Tuscan coast had emerged from the seabed, rather than sunk as suggested by Savi and Meneghini.