Travolta and Alley, who are both Scientologists, starred in the "Look Who's Talking" trilogy of big-screen comedies in the 1980s and '90s.
Travolta, who married actress Kelly Preston in 1991, has been dogged by allegations from men claiming he was sexually aggressive toward them.
"I know John. With all my heart and soul, he's not gay," Alley told interviewer Barbara Walters. "I think in some weird way, in Hollywood, if someone gets big enough and famous enough, and they're not out doing drugs and they're not womanizing, what do you say about them?"
Alley said she fell in love with Travolta when she worked with him on the first "Look Who's Talking" movie in 1989, but resisted the urge to try to make a life with him.
"Believe me, it took everything I had inside, outside, whatever, to not run off and marry John and be with John for the rest of my life," she said. "I feel like when you marry someone, you're supposed to work hard at it and you're supposed to make it work."