The story-based campaign mode consists of related scenarios with preset objectives, such as destroying a given building. In Age of Empires III, the campaign follows the fictional Black family in a series of three "Acts", which divide the story arc into three generations. All three acts are narrated by Amelia Black (Tasia Valenza).

Instead of playing as one of the standard civilizations, the player controls special civilizations linked to the character or period that each Act portrays: the Knights of St. John for Act I, John Black's Mercenaries for Act II, and the United States for Act III. Most player units speak English, with the exception of unique units such as Spanish Rodeleros and Lancers or German Uhlans and War Wagons.

Act I: Blood In the late 16th century, Morgan Black—a Scottish-born mid-level commander of the Knights of Saint John— defends their last stronghold on Malta from the forces of Sahin "The Falcon" of the Ottoman Empire (in a telling of the Great Siege of Malta). When the Turks flee, Morgan and his superior Alain Magnan discover a stone library telling of the Lake of the Moon; the Fountain of Youth, a rumored source of eternal life; and a secret society called the Circle of Ossus, who seek the Fountain for their own gain. Alain orders Morgan to sail to the New World to search for the Lake, but Morgan is attacked by the pirate Elisabet "Lizzie" Ramsey and lands in the Caribbean islands. After re-engaging and defeating Lizzie, Morgan's men find some navigation charts and sail to New Spain. In Yucatan, Morgan's forces destroy an Ottoman base. However, Spanish conquistador Francisco Juan Delgado de Leon captures Sahin. Morgan then defends his new Aztec allies from the Spanish and learns Delgado was looking for the map to the Lake of the Moon, disguised as a town square mosaic. Morgan sets sail for Florida, but his fleet is damaged by a hurricane and docks in Havana, Cuba, where he earns Lizzie's respect and promises her the gold from the Spanish treasure fleet if she takes him to Florida.

In Florida, while Alain claims the lake, Morgan and Lizzie capture Spanish treasure ships and Sahin, killing Delgado in the process. Sahin tells Morgan that he only wanted to keep the Circle from claiming the Fountain. Alain orders Morgan to execute Sahin, but Sahin and Lizzie convince Morgan that Alain actually leads the Circle of Ossus. At the Lake of the Moon, the group captures an enormous stationary cannon called a "Fixed Gun" from the Circle. Using the Fixed Gun, Lizzie's fire ships and Morgan's ground forces hold off the Circle's elite Boneguard soldiers, kill Alain, and destroy the Fountain. After the battle, Sahin returns to Turkey, and Lizzie, having lost her Spanish gold, leaves for the Caribbean, although it is hinted she and Morgan will reunite. Morgan silently wonders whether they really found the Fountain of Youth, then refills his empty canteen with water from the lake.