In Armenia, in the 3rd century, Poliuto is secretly baptized by Nearco. His wife Paolina, once betrothed to Severo, has rejected the high priest Callistene, who arouses Poliuto's jealousy by allowing him to overhear a conversation between her and Severo. The Christian Nearco is captured and condemned to death, refusing to name his most recent convert. Poliuto comes forward and is also imprisoned. In death he is joined by his wife Paolina, herself now embracing the new faith.

Donizetti reworked Poliuto for Paris as Les martyrs (The Martyrs), with a French libretto by Eugène Scribe. The French version won little success, even when translated into Italian for opera- houses in Italy, and the first performance of the original opera was given after Donizetti's death. In the tenor aria D'un alma troppo fervida (Of a soul that is too fervent), Poliuto prays for calmness of spirit, while the soprano Paolina, moved by the Christian rites, sings Di quai soavi lagrime (From these sweet tears). The baritone Severo is introduced by Di tua beltade immagine (The image of your beauty). In a final duet Paolina begs Poliuto to recant in Ah! fuggi da morte (Ah! Escape death), but soon they both declare their faith.