Barnaba, a spy for the dreaded Council of Ten, cynically observes a throng of Venetians celebrating a regatta on the Grand Canal. When the crowd rushes off to watch a boat race, he muses on his unrequited passion for the street singer La Gioconda, whom he sees leading her blind mother.
When the crowd returns, Barnaba accuses Gioconda's mother of having bewitched a boatman to make him lose the contest. As the enraged mob threatens the old woman, Gioconda returns with Enzo, a young sea captain whom she loves.
Suddenly Alvise Badoero, leader of the Ten, and his wife, Laura, appear on their way home from a masked ball. He quiets the rabble, and his wife begs mercy for the old woman. In gratitude, Gioconda's mother gives her a rosary ("Voce di donna.") Everyone goes off to church except Barnaba and Enzo, whom the spy recognizes as a banished Genoese nobleman formerly in love with Laura ("Enzo Grimaldo, Principe di Santafior, che pensi? ")
Barnaba has decided he must win Gioconda by proving her lover faithless, so he tells Enzo he can arrange for Laura to visit his ship that night. Though he distrusts Barnaba, Enzo is thrilled.
Barnaba now dictates a note to the public scribe Isépo informing Alvise of Laura's impending desertion. This he drops into the lion's mouth ("O monumento!"), the usual repository for denunciations. Gioconda has overheard, and as vespers sound, she laments Enzo's betrayal, comforted by her mother.
Disguised as a fisherman, Barnaba tosses off a barcarole ("Pescator, affonda l'esca") while waiting to deliver Laura to Enzo. When the spy leaves, Enzo arrives, extolling the beauty of sky and sea ("Cielo e mar!") Barnaba ferries in Laura.
While Enzo prepares for his elopement, Laura prays to the Virgin for protection ("Stella del marinar!") Suddenly Gioconda appears, ready to do battle for Enzo ("L'amo come il fulgor.") Laura defies her and is about to be stabbed when Gioconda sees the rosary her mother gave Laura for saving her life. Unable to harm her rival, Gioconda warns that Alvise is in hot pursuit and sends her to safety in a skiff. Rather than surrender to Alvise and the Inquisition, Enzo sets fire to his ship.
In his palace, Alvise decides to murder his faithless wife ("Sì, morir ella dé!") When she appears, he orders her to drink a vial of poison before a distant chorus has ended. But no sooner does he leave the room than Gioconda slips in to give Laura a potion to simulate her death instead.
In the ballroom, Alvise greets his guests, offering them a Dance of the Hours as entertainment. Barnaba drags in Gioconda's mother. A death knell resounds in the hall; Enzo unmasks and laments his dead beloved ("Già ti veggo,") to the wonder of Alvise's guests.
Their host orders Enzo arrested as Gioconda decides to offer herself to Barnaba if he will save Enzo. The gloating Alvise displays Laura's apparent corpse to climax his party. Enzo lunges at Alvise but is intercepted by guards.
Gioconda's friends bring the sleeping Laura to Gioconda's ruined home on the Giudecca canal; the desolate street singer resolves to kill herself ("Suicidio!") Enzo bursts in to kill Gioconda for stealing Laura's body, but Laura herself awakes, and the lovers are reunited. Overcome with gratitude, they bless Gioconda ("Sulle tue mani l'anima") and embark in a waiting boat.
Kneeling before a crucifix, worried about her mother, the girl turns to face Barnaba and keep her bargain. Stalling for time, Gioconda bedecks herself with further finery. "You wanted my body, cursed demon," she spits at him, "now take it!" Before he can move she stabs herself. Barnaba screams that he has killed her mother, but Gioconda no longer hears.