Rock Band 3 is the third game in the main Rock Band series, and features numerous new additions to the original game. As with previous games in the series, players form a band (of up to seven players) and perform a song together using guitar, bass, drums, vocals (solo or harmonies) and new to this game, keyboard. By working together, players can maximise their scores. Each player can fill a power bar by performing special sets of notes. When they have enough of these, they can activate "Overdrive" which adds a x2 multiplier for each player that has Overdrive activated simultaneously (so, for example, having four players with Overdrive activate will score you x8 rather than x2 for only one person).

Vocal harmonies, first introduced in The Beatles: Rock Band, are carried over into the main Rock Band series for the first time. Up to three players can perform on the microphone, with one player performing the lead, and the remaining players performing backing vocals. By keeping in harmony with each other, players can earn double or triple score, depending on how many players can keep up. The game also introduces a new style of play for each of the instruments, "Pro Mode". Pro Mode is more accurate to actual instruments for those players who are looking for an even greater challenge - guitars have multiple buttons on the fret board and players must actually strum strings, the drums must have the three cymbal attachment, while with the keyboard, even more keys are used so that the performance is much closer to actual play.

The Career Mode of the game has also been revised for this game. Rather than grinding to earn money to unlock new items, the game is instead based around Achievement-like "goals". Each goal you unlock earns you more fans for your band, and often unlocks new items of clothing or instruments. All songs are initially unlocked so that those who just want to play for fun can, while those looking for a deeper experience can tackle the "Road Challenge" mode, which has you travelling to different venues and performing song sets. Each star you earn for a song gets transformed into "Spades" at the end of the performance, and the more Spades you earn, the more goals you unlock which again translates into even more fans and items. Bonus Spades can be earned by performing the requested tasks at the start of each song, such as continuously deploying Overdrive, performing note streaks while you're in the spotlight, or deploying Overdrive when full and then performing note streaks.