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If there's anybody out there... anybody... please. You are not alone.
— Robert Neville's radio transmitoin

Lieutenant Colonel Robert Neville was a military virologist who self-encased himself in quarantined Manhattan to find the cure towards an infectious disease caused by a mutated measles virus that was meant to cure cancer. The disease causes its victims to become meat-seeking, pale creatures that prey on animals and surviving, uninfected humans. They were titled "darkseekers" because of their painful intoleration of UV radiation. Neville's family was tragically killed in a helicopter accident when an oncoming chopper crashed into theirs. Robert witnessed the entire ordeal and constantly receives nightmares of the event.

Neville lives in a world surrounded by overgrown wildlife and the treacherous darkseekers. His only companion is man's true best friend, his dog Sam. Due to his solitude, Neville becomes somewhat delusional and makes friends with mannequins. However, after being ensnared in his own trapped after believing his mannequin friend, Fred, moved, Neville's dog is bitten by an infected canine. Unfortunately, unlike Robert, Sam is only immune to the airborne virus, not to the contact virus.

Neville is forced to kill his dog. In a fit of rage, Neville attempts to kill himself but without killing a large number of darkseekers in order to avenge his dog's death. However, after crashing he is rescued by Ann and Ethan, two survivors. After taking Robert back to his home, Ann informs him that there is a survivor's colony, but he refuses to believe. The darkseekers raid Neville's home that night because Ann forgot to cover their tracks.

Overwhelmed, Neville, Ann, and young Ethan evacuate to the scientist's lab where he researched endlessly for a cure. He realizes that the cure lies within his blood. He extracts a sample of it and gives it to Ann to take with her when she goes to the survivor's colony. Hiding her and Ethan inside a small room, Neville takes out a hand grenade, pulls the pin, and charges toward the advancing darkseekers, resulting in a fiery explosion that eliminates the darkseekers and saves the two survivors.