A Certain Scientific Railgun is set in Academy City, a sprawling, futuristic metropolis that covers a third of western Tokyo. This city is home to 2.3 million residents, the vast majority of whom are students who have undergone the city's special curriculum to develop psychic powers, known as esper abilities. These abilities are ranked from Level 0, for those with no measurable power, to the nearly god-like Level 5, of which only seven exist in the entire city.

The story centers on one of those seven, Misaka Mikoto, the third-ranked Level 5 known as the Railgun. A student at the prestigious all-girls Tokiwadai Middle School, Misaka is an electromaster of incredible power, capable of generating and manipulating electricity to create devastating attacks, such as firing a metal coin at three times the speed of sound. Despite her immense power, she is a kind, energetic, and fiercely independent girl with a strong sense of justice. She shares a dorm room with her underclassman, Shirai Kuroko, a Level 4 teleporter who is also a member of Judgment, a student-run public safety organization that maintains order in the city. Kuroko is intensely devoted to Misaka, whom she calls sister, often to the latter's exasperation.

Kuroko's partner in Judgment is Uiharu Kazari, a gentle and cheerful girl with a talent for computer hacking, who attends the less prestigious Sakugawa Middle School. Her best friend and classmate is Saten Ruiko, an upbeat and curious girl who is a Level 0, meaning she has no discernible esper power. As a result, Saten is fascinated by urban legends circulating in the city, often hoping to find a shortcut to gaining abilities. Together, these four friends navigate the challenges of school life while becoming entangled in the darker secrets lurking beneath Academy City's shiny, high-tech surface.

The first major narrative arc revolves around an urban legend known as the Level Upper, a mysterious audio file rumored to artificially enhance a person's esper ability. When numerous students who have used the Level Upper begin falling into unexplained comas, Misaka and her friends investigate, discovering that the file connects the brainwaves of its users into a single massive network. Their investigation leads them to Kiyama Harumi, a brilliant and desperate researcher who created the Level Upper to gather enough computational power to save a group of her former students, children who were used as unwitting test subjects in a cruel experiment and have been comatose ever since.

Following this, the series adapts the Sisters arc, which was originally a pivotal storyline in its parent series, A Certain Magical Index. The narrative reveals that Misakas own DNA was stolen when she was a child and used to create over twenty thousand military-grade clones, known as the Sisters. These clones are being used as disposable targets for the citys most powerful and violent Level 5, Accelerator, in a project designed to allow him to evolve into a Level 6. Horrified and guilt-ridden, Misaka fights desperately to stop the slaughter of her clones, only to realize her powers are useless against the seemingly invincible Accelerator. At her lowest point, she receives unexpected help from a seemingly powerless boy named Kamijou Touma, whose right hand has the mysterious ability to negate any supernatural power.

The final season introduces the Daihaseisai, a massive city-wide sports festival where Misaka finds herself targeted by a sinister plot involving a scientist from her past. She is forced into an unstable and forced evolution toward a Level 6 state, which threatens to destroy the city. To save her, she must rely on the help of unexpected allies, including the mysterious Level 5 mental manipulator, Shokuhou Misaki, who has her own hidden history with Misaka. The series continues to explore the bond between the four friends, the true cost of scientific progress, and the invisible darkness that exists just beneath the surface of their seemingly ideal city.