Franz Overbeck was born in Saint Petersburg as a German citizen to Franz Heinrich Herrmann Overbeck, a German-British merchant, and his wife, Jeanne Camille Cerclet, who was born in Saint Petersburg to a French family. Consequently, his upbringing was European and humanistic: first taking place in Saint Petersburg, then in Paris from 1846 until the February Revolution of 1848, once again in Saint Petersburg, and after 1850 in Dresden. This international education helped him gain fluency in the most important European languages.
From 1856 until 1864, Overbeck studied theology in Leipzig, Göttingen, Berlin, and Jena. Primarily through the lectures of Karl Schwarz and in conjunction with the historical theology of Ferdinand Christian Baur, his studies situated him at the beginning of academic criticism against the official theology. In 1859, he received his doctorate degree, after which he worked on his Habilitation on Hippolytus until 1864. After 1864, he taught as a Privatdozent in Jena.
During his student time in Leipzig, he became close friends with Heinrich von Treitschke, and in Göttingen, Overbeck had become a member of the Burschenschaft 'Hannovera'.