"It ain't gonna happen," the presidential hopeful said at an MSNBC town hall in Buffalo, New York, on Thursday.
Host Chuck Todd asked the 45-year-old Texas senator if he would consider a Trump-Cruz ticket if the Republican National Convention urged him to do so.
The candidate held to his answer.
The statement comes just days after Trump, 69, said he would consider some of his past opponents, including Marco Rubio, John Kasich and Scott Walker, as his running mate.
"I like Marco Rubio … There are people I have in mind in terms of vice president. I just haven't told anybody names," Trump told USA Today. "I do like Marco. I do like Kasich … I like Walker actually in a lot of ways."
Last month, he told CNN's Wolf Blitzer that he has "absolutely zero interest" in running alongside the business mogul.