A secret world of creatures known as abnormals lives hidden among humans. Some are dangerous, but most are benign, becoming violent only when threatened by an encroaching world. The beautiful and enigmatic Dr. Helen Magnus, a 157-year-old English scientist, has dedicated her life to tracking these beings, providing sanctuary for the harmless while protecting humanity from the dangerous ones. She operates out of a sprawling, gothic-style facility concealed within a fictional Old City, a high-tech haven filled with extraordinary inhabitants ranging from mermaids and harpies to a gentle Neanderthal-like assistant named Bigfoot.

Joining Dr. Magnus in her quest is her fearless and impulsive daughter, Ashley Magnus, a skilled monster hunter. The team is rounded out by Henry Foss, a quirky but brilliant tech wizard and weapons designer who harbors a secret: he is a werewolf. The perspective of the audience is provided by Dr. Will Zimmerman, a skeptical and brilliant young forensic psychiatrist recruited by Magnus. Initially doubtful of the hidden world he is shown, Will uses his profiling skills to understand the abnormals' psychology, eventually becoming a crucial member of the team.

The narrative is driven by two major forces. The first is the Sanctuary team's mission to capture and rehabilitate various abnormals, such as a young boy mutated by the Chernobyl disaster, ancient Celtic warriors known as the Morrígan, or a gang of thieves who can fold their bodies to slip through impossibly small spaces. The second is their ongoing conflict with the Cabal, a powerful and shadowy organization that seeks to capture and exploit abnormals for their own sinister purposes.

A deep mythology unfolds through the backstory of "the Five," a secret group of Victorian-era scientists that included Magnus, her former fiancé John Druitt, and the physicist Nikola Tesla. After injecting themselves with pure vampire blood, each gained unique abilities: Magnus received longevity, Druitt gained the power to teleport (a power that drove him insane and led him to become the infamous Jack the Ripper), and Tesla transformed into a half-vampire. Throughout the first season, this history resurfaces as the team contends with Druitt's unpredictable nature and Tesla's dangerous schemes, culminating in a final confrontation where the Five must reunite to stop the Cabal from unleashing a biological weapon designed to turn abnormals into mindless killers.