She was married to her first cousin, the future Ferdinand III, then the titular king of Hungary, on 20 February 1631. She travelled from Spain to Austria through Italy, a dangerous journey during the Thirty Years' War, which took 14 months to complete. She was married to Ferdinand by proxy with her brother-in-law, Bishop Leopold Wilhelm of Strassburg, Passau and Halberstadt, when she reached Trieste in 26 January 1631, and to Ferdiand himself in a second ceremony in Vienna. The wedding was celebrated for a month. The marriage was described as friendly. Maria Ana was described as happy tempered, friendly and intelligent, and she was able to ease the feelings of the melancholic emperor. She was politically active, acted as the advisor of her spouse, his Regent during his absence, and followed him on his travels.
During the Thirty Years' War, the imperial family moved hastily to Linz, where she died of poisoning during her last pregnancy after fever and heavy bleeding. The child she was carrying, Maria, was still alive, and was born by cesarean section, but died soon after. Mother and daughter were buried together in a single coffin.