A genetically re-engineered measles virus, originally created as a cure for cancer, turns lethal. The virus kills 90% of the world's population and turns 9% into vampiric, albino, zombie-like, cannibalistic mutants called Darkseekers, who are extremely vulnerable to the ultraviolet rays in sunlight. The remaining 1% of the population is immune to the virus and is the prey of the mutant Darkseekers.

In 2012 three years after the outbreak, U.S. Army virologist LTC Robert Neville lives an isolated life in the deserted ruins of Manhattan, unsure if any other uninfected humans are left. Neville's daily routine includes experimenting on infected rats to find a cure for the virus, searching for food and supplies, and waiting each day for any survivors who might respond to his continuous recorded radio broadcasts, which instruct them to meet him at midday at the South Street Seaport. He also listens to the song "Three Little Birds" by Bob Marley on repeat. Flashbacks reveal that his wife, Zoe, and daughter, Marley, died in a helicopter accident during the chaotic evacuation of Manhattan as the military was enforcing a quarantine of the island. Neville himself stayed behind with other military personnel. Neville's only companion is his German Shepherd Samantha (Sam) and to cope with his loneliness, he regularly "talks" to some mannequins and the characters on film recordings from video stores. At night, he barricades himself with Sam inside his heavily fortified Washington Square Park home to hide from the Darkseekers.

One day as Neville hunts a deer, Sam pursues it into a dark building. Neville cautiously goes in after her and locates the deer's corpse along with Sam, but discovers the building is infested with Darkseekers. Both escape unharmed and the attacking Darkseekers are killed by the sunlight. Neville finds a promising treatment derived from his own blood, so he sets a snare trap and captures a female Darkseeker. A male Darkseeker attempts to pursue them but is halted by the sunlight and returns to the shadows. Back in his laboratory in the basement of his house, Neville treats the female seemingly without success. The next day, Neville notices "Fred", a mannequin usually at the local video store, positioned outside Grand Central Terminal and shoots him in confusion. However, Neville realizes he is being watched by the Darkseekers from within nearby buildings. As he approaches Fred's body, he is ensnared in a trap similar to the one he used to capture the female and is rendered unconscious after hitting his head. By the time he wakes up the sun is setting and he is attacked by infected dogs. Neville and Sam eliminate them, but Sam is bitten during the fight. Neville injects her with a strand of his serum, but when she shows signs of infection, Neville is forced to strangle her to death before she turns. Heartbroken and driven by rage, Neville ventures out and deliberately attacks a group of Darkseekers the following night, before he is rescued by a pair of immune humans, Anna and a young boy named Ethan, who have traveled from Maryland after hearing his broadcast.

They transport the injured Neville back to his home, where Anna explains that they survived the outbreak aboard a Red Cross evacuation ship from São Paulo, and are making their way to a survivors' camp in Bethel, Vermont. Neville denies this and argues that no such survivors' camp exists. Neville adjusts the potential cure and injects the female Darkseeker. The next night, a group of Darkseekers, who tracked Anna and Neville the night before, invade the house. Neville, Anna, and Ethan retreat into the basement laboratory sealing themselves in with the female test subject. Discovering that the last treatment was successful, Neville assesses the situation as the Darkseeker alpha male rams himself against a glass door to break in. Neville draws a vial of blood from the woman he cured, and gives it to Anna, before shutting Ethan and her inside a coal chute in the back of the lab. Neville then takes a grenade and kills the Darkseekers at the cost of his own life, saving the cure.

The following day, Anna and Ethan arrive at the survivors' camp in Bethel, where they are greeted by military officers and other survivors before Anna hands them the cure. Anna narrates how Neville's and Sam's efforts and sacrifices to save humanity ultimately became legend and people should take inspiration from them.