Assassin's Creed: Bloodlines is an action-adventure, stealth game set in an open world environment. The gameplay is nearly identical to that of Assassin's Creed in terms of control style, although there were minor modifications to the control scheme, due to the lack of specific keys and buttons on the PlayStation Portable. Some features from the previous game, such as Eagle Vision, were removed.

One difference between the two games is the number of civilians present on the street. While the first game was filled with random civilians, Bloodlines has little to no civilians, explained in-game that the people were in a constant state of insecurity due to the new system of governance. Still, civilians will react to Altaïr's improper behavior, such as scaling buildings, and running around the streets.

Stealth in Bloodlines was downgraded due to the control scheme. Blending was also downgraded, due to the system platform. Although scholars are still present in-game, Altaïr can no longer use them as a way to hide. Although Altaïr can still blend, it only acts as a way to safely bypass guards without raising suspicion. Scaling the side of buildings was made easier and faster, resulting in simpler animations, again, due to the system's inferiority. High and Low profile movements are still present in-game, though they have been downgraded as well.

Taking the place of flags, a new collectible featured in the game are Templar Coins that can be used to upgrade the player's health bar, and the amount of damage a weapon can inflict. Three types of coins are present in game: Bronze, Silver and Gold, with Gold holding the highest value. Similar to the previous game, side-missions are available to players, such as saving civilians from Templar soldiers and performing specific tasks for allies of the resistance. The variety of tasks has been expanded, such as delivering letters, and intercepting couriers. In return, Altaïr will receive coins as a reward, instead of assistance from previous groups, such as scholars and vigilantes.

The game is primarily set on the island of Cyprus, where players can freely explore the cities of Limassol and Kyrenia. Due to the smaller size of the map, horseback riding was removed in Bloodlines. Like in the previous game, Altaïr can synchronize from high vantage points around the city in order to map out the city. Although the map itself is already drawn out, synchronizing will provide the locations of various side-missions.

Bloodlines features a few new assassination techniques and weapons. Weapons from the previous game are also present, such as Altaïr's sword, his hidden blade, throwing knives, and his fists. Assassinations are also similar to the first game, although the animation has been toned down to better fit the system. A new, yet very minor assassination technique has been added to the game, allowing Altaïr to pull opponents off of ledges, similar to Assassin's Creed II.

In Bloodlines, cut-scenes are played through preset dialogue, with the character models acting in preset motions and gestures. Unique gestures were only present in confrontations between boss characters, such as Moloch and the Dark Oracle. Subtitles were also added to the game. This enabled easier understanding of the game plot

Story missions and assassinations were simplified and straightforward. As a replacement for eaves-dropping and pick-pocketing, missions are structured similarly to Assassin's Creed II, with Altaïr handling one task after another and slowly gathering vital information from both his allies and enemies as each mission is completed.

Bloodlines features in-game achievements, which reward players with Templar coins. Achievements can be earned after performing specific tasks, such as killing a set number of soldiers in a specified order, and also collecting all of the Templar coins located throughout the game world.

Despite all the downgrades, the game has a unique new feature, not present in any other Assassin's Creed title. Altaïr can now regain throwing knives by walking over his used ones. Since pick-pocketing was removed and was the only way to regain throwing knives in the first game, Altaïr can now re-use his throwing knives provided that: the knife he threw landed on the ground or an enemy; if the knife lands on water, Altaïr cannot retrieve them. Throwing knives may also be regained by interacting with the newly added knife box in the safehouses.