Born in Krauchenwies Castle in Krauchenwies, Sigmaringen, in 1837, Stephanie was the eldest daughter of Prince Karl Anton of Hohenzollern, head of the House of Hohenzollern-Sigmaringen, and his wife Princess Josephine of Baden.

Her maternal grandparents were Karl, Grand Duke of Baden, and Stéphanie de Beauharnais, adopted daughter of Napoleon.

Stephanie married King Peter V of Portugal by proxy on 29 April 1858 at St. Hedwig's Cathedral in Berlin, where her eldest brother Leopold stood in for the groom.

Stephanie fell ill with diphtheria and died only a year later in Lisbon at the age of 22.

Her body was interred at the Braganza Pantheon inside the Monastery of São Vicente de Fora in Lisbon.