Picking up a few weeks after where Ben 10: Alien Force leaves off, Ben 10 Ultimate Alien follows the now sixteen-year-old Ben. The Omnitrix has been destroyed and Ben must learn to master the secrets of the new Ultimatrix , a gauntlet-like version of the old Omnitrix that not only gives him access to all of his original powers and abilities, but also allows him to evolve his alien forms into even stronger and even more powerful versions (called "ultimate forms"). In the premiere episode "Fame", a young ten-year-old fan named Jimmy Jones deduces Ben's secret identity and reveals it to the world. Ben becomes an overnight celebrity, hailed by kids as a hero but distrusted by most adults as a possible threat. With Gwen, Kevin and Grandpa Max still at his side, Ben continues to battle alien threats to the galaxy including finding five aliens that were attacked by a villain named Agreggor. After Agreggor captured and fused with all five aliens, he began searching for the Map of Infinity. Later Kevin becomes once again a major antagonist to the Ben 10 series after he absorbs energy from the Ultimatrix and Aggregor and goes insane with power but is shown that he does still have a mind and still does care and that he has not gone completely insane.
- Benjamin "Ben" Kirby Tennyson (voiced by Yuri Lowenthal) - At sixteen years old, Ben now wields the incredibly powerful Ultimatrix gifted to him by Azmuth. The device that allows his aliens forms to evole into more stronger versions of themselves, which aids him against more intense foes where his normal transformations are either too weak or inadequate. His secret identity has been revealed by a boy named Jimmy Jones that has spread to the world his secret, earning him a mix of adoration and suspicion, mostly from kids and adults, respectively.
- Gwendolyn "Gwen" Tennyson (voiced by Ashley Johnson) - At sixteen years old, Gwen is still the level-headed one of the superpowered trio. Continuing to refine the use of both her Anodite and magical powers and abilities which both appear to have greatly strengthened and amplified enormously over the past year, Gwen is shown to be a valued member of the team.
- Kevin Ethan Levin (voiced by Greg Cipes) - At seventeen years old, Kevin continues to be the arrogant and trouble-making member of the group. Despite questionable antics, Kevin excels much more often, proving to be a true and loyal friend now that he has learned from his past mistake of absorbing energy (which causes Osmosian's to go insane). After his Omnitrix mutation was cured, Kevin regained his original Osmosian powers and abilities to absorb all types of energy and matter. He can copy any matter he absorbs into an armor. Moreover, learning from his experience, Kevin is also able to temporarily absorb extra mass to greatly strengthen and augment his powers and abilities even further and shapeshift his limbs into makeshift tools and weapons. He can also morph or fuse together the object he absorbed, and he is shown to be able to temporarily let a subject become any matter he absorbs as long as he remains in contact with the subject. He now owns a modified Plumber spaceship in addition to his car. In the episode "The Forge of Creation", he absorbed the energy of the Ultimatrix to stop Aggregor and turned into a grotesque amalgam once again. As a side effect of the energy absorption, he is driven insane, though, unlike the original series, he is more of an anti-villain than a true villain, as his good heart has not been entirely suppressed by his self-induced insanity.
- Julie Yamamoto (voiced by Vyvan Pham) - At sixteen years old, Julie begins to show more interest in the secret identities of Ben and Gwen. She encourages him not to hide from society even though most adults see him as a problem.
- Aggregor (voiced by John DiMaggio) - A mysterious Osmosian who kidnapped five aliens (Bivalvan, Andreas, P'andor, Ra'ad and Galapagus) from the Andromeda Galaxy, who then escaped from his ship to Earth. Now he is hunting them down one by one, intent on draining their powers to obtain what he calls "the ultimate prize" which eventually turns him into Ultimate Aggregor. Aggregor currently serves as the main antagonist of the series, searching for the Map of Infinity which can give the user its power to travel anywhere in seventeen dimensions. He is later stopped by Ultimate Kevin very easily, and Aggregor is unconscious.