"The president's speech left the impression that he believes reforming immigration quickly is more important than reforming immigration right," the Florida Republican said in a statement.
He expressed concern Obama's approach, outlined Tuesday in a Las Vegas speech, did not link border security with giving illegal immigrants a way to become citizens.
"Without such triggers in place, enforcement systems will never be implemented and we will be back in just a few years dealing with millions of new undocumented people in our country," Rubio said.
At the same time, 41-year-old Rubio, a potential 2016 presidential candidate, pushed back against conservative critics who questioned a bipartisan framework he and seven other senators unveiled Monday.
He defended the blueprint to conservative radio talk show host Rush Limbaugh, emphasizing the senators' proposal would make citizenship contingent on stricter enforcement rules.
"If there is not language in this bill that guarantees nothing else will happen unless these enforcement mechanisms are in place, I won't support it," Rubio said.
Limbaugh eventually said he saw the reform's benefits.
"What you are doing is admirable and noteworthy," Limbaugh told Rubio, even as he warned the first-term senator against compromising with Obama.
"You are recognizing reality. You're trumpeting it, you're shouting it," Limbaugh said. "My concern is the president wants to change the reality."
Sen. Lindsey Graham, R-S.C., also challenged those criticizing the senators' reform efforts.
"To all the people who don't like what we're doing, come up with a solution yourself and see how much support you can get for it," the Los Angeles Times quoted him as saying.
"If you're willing to accept the status quo, and all you're going to do is complain, then you're the biggest author of amnesty, not me," Graham said, referring to a de facto amnesty already happening because immigration rules are not enforced.
Sen. John McCain, R-Ariz., Obama's 2008 opponent, said in a statement he appreciated Obama's support for the senators' efforts in his Las Vegas remarks.
"While there are some differences in our approaches to this issue, we share the belief that any reform must recognize America as a nation of laws and a nation of immigrants," said McCain, who led a failed 2007 immigration-overhaul effort before focusing on toughening U.S. border security during his 2010 re-election campaign.
"We should all agree that border security and enforcement is particularly important in order to ensure that we don't repeat the mistakes of the 1986 immigration reform," he said in a statement. "The road ahead will be not be easy, but I am cautiously optimistic that working together, we can find common ground and move forward on this vitally important issue."
House Speaker John Boehner, R-Ohio, said in a statement through a spokesman: "There are a lot of ideas about how best to fix our broken immigration system. We hope the president is careful not to drag the debate to the left and ultimately disrupt the difficult work that is ahead in the House and Senate."