Act I: The Lion’s Jaws
In the courtyard of the Doge's Palace in Venice, the people celebrate the annual regatta. Barnaba, a spy and former informant of the Inquisition, watches over the festivities. He is filled with desire when he sees the beautiful singer La Gioconda guiding her blind mother, La Cieca, across the courtyard. La Gioconda leaves her mother to go in search of her beloved Enzo in the crowd, and Barnaba chases after her. When he is sharply rejected by her, Barnaba imagines getting revenge by attacking La Gioconda’s failing mother.
The people jubilantly welcome the winner of the regatta. One of the losers, Zuàne, has difficulty accepting his defeat. Barnaba whispers that he owes his defeat to a sinister spell cast by La Cieca. Hearing these words, Zuàne's supporters shout that La Cieca is a witch and that she should be executed. As Barnaba is about to have her arrested, Enzo tries to defend her. The young man is in conflict with the people who demand loud and clear that the old woman be lynched.
Alvise Badoero, one of the Inquisition's leaders, and his masked wife, Laura Adorno, come to see the cause of the disturbance. Barnaba urges them to sentence La Cieca, but Laura, moved by La Gioconda's pleas, manages to convince her husband to acquit her. In gratitude, La Cieca offers her rosary to Laura.
Barnaba makes Enzo understand that he holds him at his mercy: the spy knows that the young man is the outcast Enzo Grimaldo who has returned to Venice in secret, and that his relationship with La Gioconda is only to disguise his true love for Laura.
Once Enzo has gone to prepare for his rendez-vous with Laura that night, Barnaba dictates an anonymous letter to Alvise telling him of Laura's plan to escape with the outcast. Barnaba places the letter in the jaws of a post box shaped like a lion in which Venetians can slip their anonymous denunciations of offenders against the law. La Gioconda, who has left the church, listens unnoticed and witnesses Barnaba's revelation of Enzo's true love. Her heart is broken, while Barnaba is happy: as a spy, he considers himself more powerful than the doge.
Act II: The Rosary
Enzo waits aboard his boat while the crew prepares for departure. Just as the outcast believes Laura must no longer be coming as planned, she is led to the boat by Barnaba. The lovers embrace each other and dream of their new life together.
La Gioconda comes out from her hiding place on the boat and is unable to repress her jealousy and hatred towards Laura. The singer thinks of stabbing her but, seeing Alvise approaching on another boat, decides to let Laura face her husband’s revenge instead. In despair, La Gioconda grabs her rival’s rosary. She recognises it as La Cieca's and realises that Laura is the one who saved her mother from death. To repay the debt, the singer lets Laura escape by making her own boat available to her.
Enzo notices Laura's disappearance and finds himself facing La Gioconda. She points out to him the Venetian galleys that are surrounding his boat. In desperation, the outcast sets fire to his boat and swims away.
Act III: The Golden House
While a ball gets under way in the Ca' d'Oro, one of the richest palaces along the Grand Canal of Venice, Alvise plots revenge against his wife. He decides to make her drink poison, effectively forcing her to commit suicide and so condemning herself to hell. He shows Laura a catafalque that waits to receive her and he hands her a vial of poison to drink before the end of the ball.
La Gioconda has found her way into the palace and finds Laura alone just as she is about to ingest the poison. The singer exchanges her rival’s deadly beverage with a powerful drug that will plunge the young woman into a deep sleep that gives the illusion of death.
Checking up on his wife, Alvise finds Laura's body on the catafalque next to the empty vial. Satisfied to have been avenged, he welcomes his guests to the to ball, among them Enzo in disguise and Barnaba. Lavish entertainment is provided, including a sumptuous ballet, The Dance of the Hours. The mood of revelry is shattered as a funeral bell begins to toll and Laura still body is revealed. A distraught Enzo throws off his disguise and is promptly seized by Alvise's men.
Act IV: The Orfano Canal
Still asleep, Laura is secretly brought into La Gioconda's house by two men. She asks them to go in search of her mother, whom she had not seen since her arrest by Barnaba. The singer is torn between on the one hand her decision to reunite Laura and Enzo and then commit suicide, and on the other hand the temptation to return to Enzo and leave Laura to fend for herself. La Gioconda’s thoughts are interrupted by the cry of a gondolier who claims to have found a body in the Orfano Canal, and by the arrival of Enzo, whom Barnaba had liberated.
La Gioconda admits to her former lover that she had Laura's body collected. Enzo becomes violent towards the singer for letting him believe that Laura was dead. Just as he is about to kill her, Laura wakes up and reveals to Enzo that is was her rival who saved her from her husband. La Gioconda has also organised the couple’s escape from her house: a boat arrives to pick up Laura and Enzo and take them to safety.
Left on her own, La Gioconda fearfully waits for Barnaba to come and take his ‘payment’ for freeing Enzo. In a panic, she tries to escape but is caught by the spy. La Gioconda promises to give him her body as agreed and, drawing a dagger, she stabs herself to death. In his rage, Barnaba has the last laugh: he shouts to the dying women that last night he drowned her mother.