Assassin's Creed II is an action-adventure game played from a third-person perspective and set in an open world based on late 15th-century Renaissance Italy. Gameplay is nonlinear, and focuses on hack and slash combat, stealth, and exploration. The Animus 2.0, a new version of the machine of the same name present in Assassin's Creed, 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 doctor or use of medicine (which can be purchased from doctors or found on bodies).
Assassin's Creed II features several new means of transport, such as gondola boats.
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 Leonardo da Vinci is present in the game, aiding the player by creating new weapons from translated "Codex pages" that Altaïr, the original game's main character, left behind for future Assassins' analysis and insight. Leonardo's flying machine (based on real-life aeroplanes) can also be used during one mission. Other means of transport available in the game include horses, which can only be ridden outside of the major cities; gondolas, found most prominently in Venice; and a carriage, which is also ridden during a mission involving Leonardo. Each of the places the player can visit is more detailed compared to Assassin's Creed, and NPCs have a more human-like behavior; for example, civilians can sometimes be caught coughing or sneezing. Additionally, the player can hire groups of mercenaries, courtesans, or thieves to fight, distract, or lure guards, respectively. A day and night cycle has been added to the game, giving the game more of a sense of time, in addition to setting missions and events at certain times of the day. 
There are many ways to interact with NPCs. Money can be thrown to the ground, or a corpse carried and then deposited on the ground, may also serve as a distraction for both guards and peasants. There are also several different types of enemies, some more agile or stronger than others, and some of whom will actively search hiding places where Ezio was last seen. The player can also mock an enemy during combat.
The combat system is more complex than that of its predecessor, with the ability to disarm opponents using counter-attacks while unarmed. If the player steals an enemy's weapon, it is possible to follow up with an attack that instantly kills the enemy. Leonardo provides the player with specialized weaponry, such as dual hidden blades, poison blades, and the hidden gun (a miniature wheellock firearm), which are all based on schematics found in Altaïr's Codex pages. Generic swords, cutlasses, maces, axes, and daggers can all be purchased from vendors in each city. The player can also pick up any weapon on the ground or use improvised weapons, such as brooms or halves of a spear. In addition, players are able to purchase artwork for their villa, obtain new armor as the game progresses, and even dye Ezio's clothing with a number of different colors. Other equipment includes larger pouches to carry more throwing knives and medicine. Six additional weapons can be unlocked in the PlayStation 3 version of the game by connecting the console to a PlayStation Portable which contains a copy of Assassin's Creed: Bloodlines. 
The Auditore family's countryside villa, located in Monteriggioni, acts as Ezio's personal headquarters. The surrounding property, which includes vendors selling items such as medicine, weapons, and clothing dyes, can be upgraded with earned florins, granting Ezio discounts at the shops and increasing the passive income that is generated by the villa over time. This income is deposited in a chest inside the villa, and must be collected regularly. Purchasing upgrades for Ezio and paintings also increases the villa's overall worth.
There is now a broader array of methods for hiding or blending in the area. One can dive underwater to break guards' line of sight, and blending may be performed with any group of people, rather than only a specific type (as in the first Assassin's Creed). The game features a notoriety system, with guards more alert to Ezio's presence depending on his behavior, location, and current mission. This infamy can be reduced through bribery, removing wanted posters, or assassinating corrupt officials. 
The missions in the game now have an expanded variety, with different structuring. For example, a mission may have the objective to escort someone but may change to a chase and assassination. An investigation is less explicit, and instead, missions may follow people and/or a narrative. There are roughly 150 missions in the game, two thirds of which are part of the main storyline, while the rest are optional side quests, like assassination contracts and races. Cities also contain hidden locations such as catacombs and caves (the design of which have been compared by the developers to the Prince of Persia series), where the objective is to navigate the area. Exploring these locations eventually rewards the player with an Assassin's Seal; the collection of all six Seals allows the player to unlock the Armor of Altaïr, which is stored in a concealed section of the Villa.