Players directly control the on-screen character through a third-person perspective and can control the camera, allowing for a 360° view of their surroundings. The game takes place in an environment with , allowing the player to roam freely within several regions of late fifteenth-century Italy such as , , , , , ( only) and the countryside. The Animus 2.0, a new version of the machine of the same name present in Assassin's Crreed, provides in-game context for changes and additions to several game elements. A database is also available, providing extra historical information about key landmarks, characters, and services that the player encounters. The health system has been made more dynamic, with synchronization to the Animus and causing the character to recover only from minor injuries. More grievous injuries require visiting a street-side or use of medicine (which can be purchased from doctors or found on bodies).
The player may now swim in water, and Eagle Vision—the ability to identify specific people and landmarks—can now be used in third-person view and while moving. A young is present in the game, aiding the player by creating new equipment from translated "Codex pages" that Altaïr, the original game's main character, left behind for future Assassins' analysis and insight. Within the game, the player will be able to use Leonardo's (based on real-life plans by Leonardo) during one mission. The player also has the ability to control a carriage in one level, and can row , as well as ride horses at any point in the game where they are readily available between towns and cities. The setting of the various places the player may go to have been made more detailed and in-depth; civilians sometimes cough or sneeze. Additionally
- The combat system is more complex than that of its predecessor
- There are many ways to interact with NPCs. Money can be thrown to the ground,
- The Auditore family's countryside villa, located in , acts as Ezio's headquarters
- There are several outlets for using currency, with vendors selling items