Dom Sebastian plans a crusade against the Moors, while the Grand Inquisitor plots to make use of his absence to subvert his power. The King is accompanied by the poet Camoëns and by Zayda, whom he has rescued from the Inquisition and intends to return to her father. At home her father insists that she marry a Moorish chief, but she is in love with Sebastian and after his defeat by her proposed husband finds him wounded on the battlefield. She offers freely to marry the chief, Abayaldos, if he will spare the life of the wounded man, whose identity he does not suspect. In Lisbon once more, where the regent Dom Antonio has declared himself king, Sebastian reveals his identity, but is imprisoned, together with Zayda. He signs, as required, an instrument of abdication, in order to secure her release, but both of them are killed as she tries to aid his escape.

The last of Donizetti's operas, Dom Sébastien, roi de Portugal, has a complex and cumbersome plot.

In the concert-hall the tenor Seul sur la terre (Alone on the earth) and baritone O Lisbone, o ma patrie (O Lisbon, O my country) have a secure place.