Caroline was born at Biebrich, Nassau-Usingen the second child and first daughter of Karl Wilhelm, Prince of Nassau-Usingen (1735–1803), and his wife, Countess Caroline Felizitas of Leiningen-Dagsburg (1734–1810), daughter of Christian Karl Reinhard, Count of Leiningen-Dagsburg-Heidesheim.

Caroline, via her mother's roots among the Alsace lords of Leiningen, was a cousin of the Danish aristocrat, the Duke of Augustenborg, as both were direct descendants of the important Danish and Sleswicker magnate and statesman Frederik Ahlefeldt.

Caroline married on 2 December 1786 in Biebrich to Landgrave Frederick of Hesse-Kassel (1747–1837), youngest child of Frederick II, Landgrave of Hesse-Kassel and Princess Mary of Great Britain, daughter of George II of Great Britain.

[1] The Landgrave and Prince Frederik was born a German (Hessian) aristocrat, a cadet son of a Landesfürstliche house, but had lived since his youth in Denmark, as had his two elder brothers.

Frederik's two elder brothers married daughters of the deceased King of Norway and Denmark.