Set on Cimarron Street in 1976 Los Angeles after an apocalyptic war that ravages the land with weekly dust storms, the novel details the life of Robert Neville in the months and eventually years after the outbreak of a pandemic that has killed the rest of the human population and turned infected survivors into "vampires". The vampires conform remarkably to their stereotypes in fiction and folklore: they are blood-sucking, pale-skinned, and nocturnal, though otherwise indistinguishable from normal humans. Neville, possibly the sole survivor of the pandemic, barricades himself indoors nightly as swarms of vampires violently surround his house. He is further protected by the traditional vampire repellents of garlic, mirrors, and crucifixes. During the day, the vampires are inactive, allowing Neville to drive around stabbing them with wooden stakes (since they seem impervious to his gun’s bullets), which causes them to instantly liquefy, and scavenging for supplies. Occasional flashbacks reveal the horrors of how the disease claimed the lives of his wife and daughter.
Suffering from extreme isolation, depression, and alcoholism, Neville determines there must be some scientific reasons behind the vampires' origins, behaviors, and oddly specific aversions, so he gradually researches at his local library, discovering that the root of the disease is probably a Bacillus strain of bacteria capable of infecting both living and deceased ("undead") hosts. His experiments with microscopes also reveal that the bacteria are deadly sensitive to garlic and sunlight. After he painstakingly attempts to win the trust of a stray sickly dog that dies after only a week, Neville, heartbroken, commits himself even more vigorously to his studies. Soon he experiments directly on incapacitated vampires, which leads to a new theory that vampires are affected by mirrors and crosses because of "hysterical blindness", in which the infected now delusionally react as they believe they should when confronted with these items. Neville additionally discovers that exposing vampires to direct sunlight or inflicting wide oxygen-exposing wounds causes the bacteria to switch from being anaerobic symbionts to aerobic parasites, rapidly consuming their hosts when exposed to air and thus giving them the appearance of instantly liquefying. However, he discovers the bacteria also produce resilient "body glue" that instantaneously seals blunt or narrow wounds, explaining how the vampires are bulletproof. Lastly, he deduces now that there are in fact two differently-reacting types of vampires: conscious ones who are living with a worsening infection and undead ones who have died but been partly reanimated by the bacteria.
After three years, Neville suddenly sees a terrified woman named Ruth in broad daylight. The two cautiously gain each other's trust and even share a romantic embrace. Neville explains some of his findings, including his theory that he developed immunity against the infection after being bitten by an infected vampire bat years ago. He prepares to test Ruth to determine if she is infected or immune, vowing to treat her if she is infected, but she knocks him unconscious. Once Neville comes to, he discovers a note from her confessing that she is indeed a vampire herself. Her note suggests that only the undead vampires are pathologically violent but not those, like her, who were alive at the time of infection and who still survive due to chance mutations. These living-infected have slowly overcome their disease and are gradually developing a new society and new medications. Ruth admits she was sent to spy on him by her comrades and that he was responsible for the deaths of many of her fellow vampires, including her husband. Still, Ruth reiterates her romantic feelings for Neville and urges him to flee the city to avoid capture.
Neville ignores Ruth's warning, assuming he will be treated fairly by the new society of living-infected. However, his mind is changed when he watches a group of them annihilate the undead vampires outside his home with fiendish glee. In a panic, Neville opens fire on them but is in turn shot and subdued. Imprisoned and dying, he is visited by Ruth, who informs him that she is a senior member of the new society but, unlike the others who perceive him as a murderer, she does not resent him. She acknowledges the public need for Neville's execution but, out of mercy, gives him a packet of fast-acting suicide pills. Neville accepts his fate and asks Ruth not to let this society become too heartless. Ruth promises to try, kisses him, and leaves. Neville goes to his prison window and sees the infected staring back at him with the same hatred and fear that he once felt for them. He realizes that he, a remnant of old humanity, is now a legend to the new race born of the infection. He acknowledges that their desire to kill him, after he has killed so many of their loved ones, is not something he can condemn. As the pills take effect, he is amused by the thought that he will become their new superstition and legend, just as vampires once were to humans.
