As a spinoff, Brotherhood of Steel's gameplay greatly differs from that of other Fallout games. The gameplay is linear, not open-world. Instead of being able to travel freely across a broad world full of places and events as in other Fallout games, the player is confined to one location at a time. Previously visited locations cannot be visited again, and new locations can only be discovered by advancing the story. There are 50 distinct zones of varying size in Brotherhood of Steel.

Brotherhood of Steel uses many of the same mechanics as other entries in the Fallout series, including the SPECIAL (Strength, Perception, Endurance, Charisma, Intelligence, Agility, Luck) attribute assignment system. These seven attributes are assigned numeric values and govern most interactions in the game. Unlike other Fallout games, however, the values are constant for each character rather than customizable.

Skills exist in this game, but they do not behave like skills in other Fallout games. Rather, they are analogous to perks. When the player levels up, they receive "skill points" to increase the power of skills. Some skills also have level restrictions.

Immediately prior to Brotherhood of Steel, the three playable characters (Cain, Cyrus, and Nadia) have joined the Brotherhood as new Initiates. After selecting which character to play as (hereafter referred to as the Initiate), the game opens with the Initiate searching for missing Paladins in the nearby town of Carbon.