Educated at Leipzig and then at Wittenberg, where he became a theology professor in 1511, Amsdorf attended the Leipzig debate with Luther in 1519 and the Diet of Worms two years later, where he participated in the plan to protect Luther from his detractors by pretending to kidnap him while actually lodging him secretly in Wartburg, a castle near Eisenach. Amsdorf subsequently aided the Reformation at Magdeburg (1524), Goslar (1531), Einbeck (1534), and Schmalkalden (1537). When Luther in 1539 supported the intended bigamous marriage of Philip, landgrave of Hesse, Amsdorf opposed him. He was made bishop of Naumburg in 1541.

As a Reformer holding a bishopric, Amsdorf found his position difficult but remained under Luther's persuasion until it was necessary to yield to the Roman Catholic bishop Julius von Pflug in 1547. Exiled to Magdeburg, Amsdorf superintended the Jena edition of Luther's works. Continuing to fight for the purity of Luther's doctrine, he opposed the views of Philipp Melanchthon (Melanchthon, Philipp) and other Reformers. In particular he emphasized that salvation could come only to persons of faith and that their efforts to perform good works might even be self-defeating. Among Amsdorf's extant writings are numerous letters and varied short works.