Assassin's Creed II takes place in an open world with nonlinear gameplay, allowing the player to free roam within the four regions: the cities of Venice, Rome, and Florence, and the Tuscan countryside; acquiring weapons which the character may buy from vendors at will. Also available is colored dye used to change the color of the player-character's outfit. An article published in Game Informer revealed that Assassin's Creed II will grant the player the ability to swim and use the canals of Venice as hiding spots and will allow the player to drag city guards into the water as a means of assassination. At E3 2009, it was confirmed by the creative director that Ezio will be able to "drive small boats". A young Leonardo da Vinci is present in the game, as an aid to the player who provides a host of weapons and contraptions available for use. At one point in the game, the player can access da Vinci's flying machine which he actually built, keeping airborne using thermals from fires lit by friends gathered previously in the game. The game will feature a host of new mission types for the player to complete.

ScrewAttack revealed that there will be many weapons included in the game such as halberds, axes, long pikes, swords, daggers, and numerous others. Six additional weapons can be unlocked by players who completed Assassin's Creed: Bloodlines by linking their PSP to their PS3. Additionally, the player can disarm opponents and use their own weapons against them. The game is stated to have a much more sophisticated combat system; the player will have to use a variety of tactics on different enemies and - as demonstrated in E3 2009 demo - can now assassinate two people at once by means of Ezio's dual hidden blades. The player will also be able to automatically commit a stealth kill if the player is positioned correctly in proximity to the target and if Ezio remains anonymous without having to select the hidden blades. The poison blade is another one of the hidden blade upgrades that Ezio will get from da Vinci.

The developers said that while in Assassin's Creed there were around 5-6 mission types, there are around 14-16 mission types in Assassin's Creed II. This makes for bigger missions altogether, as a mission may start as an escort mission, then become a chase mission, and then end as an assassination mission. The structure of missions from Assassin's Creed is taken away, so there is no more investigating, as missions are available from people on the street and people met in the game. There are roughly 200 missions in this game; one hundred are part of the main narrative, while the other hundred are side quests.

The health system has been adjusted to allow for a more dynamic and participatory experience, meaning that the "memory sync" system used in the first game only helps to recharge health up to a certain point. More grievous injuries will require a trip to one of the many street-side doctors, who can help return Ezio to full strength.

As well as using water as a hiding place, the player can utilize a broader array of scenery as cover and can now blend in with any crowd rather than a group of scholars dressed in white, as in Assassin's Creed. Like in the 2006 video game Hitman: Blood Money, there will be a notoriety system, and the player will become more notorious as Ezio completes more missions, however Ezio's infamy can be reduced by bribing, removing wanted posters of him or assassinating witnesses. Also, a day and night cycle has been added to the game, giving the sense of a passage of time but additionally certain missions will only be able to be performed during the day or night . The game will feature some sort of economic system that makes it possible to hire services from non-player characters (NPCs). Ezio can steal from any of the NPCs, and likewise there are NPCs who can also steal from Ezio. The player can also use money to buy tools made by Leonardo da Vinci.

Besides the main cities of the game, the player can access hidden locations such as catacombs and caves, the contents of which have been compared by the developers to the Prince of Persia series. Exploring these locations rewards the player. It has also been said that the player can choose to throw money to the ground, which attracts the attention of nearby civilians, creating a distraction. Additionally, the eagle vision from the first game has been improved, now giving the option of remaining in third person with eagle vision initiated, allowing the player to spot targets while moving.

The animus has also been updated in Assassin's Creed II. This time, it will have a database, giving extra information about all the key locations, characters and services that the player will encounter during play. For example, when the player approaches a famous church - like Santa Maria Novella in Florence - with a simple button press, its history is revealed.

The trailer, which debuted at E3 2009, displayed many new things such as improved parkour mechanics. The main reveal of the trailer involved Ezio withdrawing his hidden blade to access a rudimentary gunpowder-based mechanic on his wristblade to take out a target from a distance. It has also been confirmed at E3 2009 that Desmond himself will "do more than just walk" in this game. Another feature, as seen in the gameplay video, no cover is safe, as guards can search them. To counter this Ezio has the ability to assassinate the incoming guards before detection, and placing the bodies in his former hiding location (he drags them into haystacks, places dead enemies on the bench he was sitting at between two allies etc). Another aspect revealed in the gameplay trailer was the addition of "smoke bombs" to aid in Ezio's escape.

Another new feature in Assassin's Creed II is the economy system which allows players to use an in-game currency to purchase items such as poison bottles, armor and swords. Players may also hire mercenaries and women to distract guards.