Gameplay-wise, Serious Sam: Xbox is essentially the same as both Serious Sam: The First Encounter and Serious Sam: The Second Encounter, though there have been many changes and modifications. There are also several features which are otherwise not present in Serious Sam 1.
For instance, there is a "multikill" system, which multiplies your score if you kill multiple enemies with a single shot. (x1 multiplier for each enemy killed) The more multikills you do in a short period of time, the higher the multiplier. Other features include a Life system, which re-spawns you in place when you die, and auto-aiming. The player can access multiplayer via System Link and split-screen.
The player can also align their favorite weapons to the X and Y button by selecting a weapon and holding X or Y, then press X or Y again to select that weapon.
- Sam now has a new model, and no shades. He is a lot less like Duke Nukem, like he was in Serious Sam 1.
- Rocketeers, Bombers, Firecrackers and Kamikazes look more muscular than in Serious Sam 1, and they have less human-like heads (except Kamikazes, who have no heads), without a mouth and with glowing light blue eyes and they all have a belt with a skull on it. The Kamikaze's back also looks different.
- Cucurbitos have fingers and a different chainsaw. Ugh-Zan III also has fingers.
- Some levels are split into two parts, such as:
- Karnak is split up into "Karnak" and "Ankh Pool".
- Sierra de Chiapas is split up into "Sierra de Chiapas" and "Catacombs".
- The City of the Gods is split up into "The City of the Gods" and "Inner Sanctum".
- Ziggurat is split up into "Ziggurat" and "Gates of Persepolis".
- The Courtyards of Gilgamesh are split into "Courtyards of Gilgamesh" and "Temple of Gilgamesh".
- Tower of Babel is split up into "Ceremonial Halls" and "Tower of Babel".
- The Citadel is split up into "The Bridge" and "The Citadel".
- Land of the Damned is split up into "Winter Wonderland" and "Lava Caves".
- The Grand Cathedral is split up into "Corridor of Death" and "The Grand Cathedral".
- Deathmatch maps Compound, Desert Temple, Red Station and We Got Skulls'n'Bones Too from the PC version have been removed. They have been replaced by 5 new maps: Ashes to Ashes, Brkeen Chevap, Docking Bay, Hoodlum's Backyard and Sun Palace.
- There are some new cinematics, and some different cinematics, such as the intro and The Second Encounter intro is skipped to where Sam flies aboard the SSS Centerprice. Specifically, Oasis, Alley of the Sphinxes, The City of the Gods, Ziggurat, The Citadel, Land of the Damned and The Grand Cathedral have new intros, while the Temple of Hatshepsut, Tomb of Ramses III, Valley of the Kings, Dunes, Suburbs, Sewers, Metropolis, Karnak (or "Ankh Pool", here), Luxor, The Pit, Tower of Babel and The Citadel had new outros.
- There is a time-lock in the Ziggurat and The Citadel, which serve as "exit" time locks. "Entry" time-locks look like doorways.
- The Military Knife, Schofield .45, Double Barrel Coach Gun, Thompson submachine gun and MK III Grenade Launcher have different models. The Serious Bomb also has a model, and is required to be equipped first, then used, unlike Serious Sam 1, where it is instantly used.
- There are Serious Bombs scattered around The First Encounter levels, and there's a P-LAH Chainsaw in Temple of Hatshepsut.
- The player can pick up ammo, health and armor items even if you have full ammo for that weapon, full health and full armor, unlike in the PC version.