"I can't imagine how it could happen, so no," Ryan said in an interview with CBS' "60 Minutes," when asked if he would support Trump's proposal.
The Republican frontrunner's plan is influenced by President Dwight Eisenhower's controversial campaign that deported nearly 1 million undocumented Mexican immigrants in the 1950s, and has been criticized by Democrats and Republicans alike.
The Wisconsin Republican's own immigration position has come under scrutiny since he decided to pursue the top job in the House last month.
Ryan, who has previously called for comprehensive immigration reform in 2013, said Sunday he still personally supports a pathway to legal status for undocumented immigrants "as a way to make amends with the law."
"You need to have a vibrant legal immigration system," he said. "Legal immigration is America. My name is 'Ryan.'"
As speaker, he's said he won't work with President Obama on comprehensive reform -- arguing that the president has proven "untrustworthy" by trying to go around Congress with executive actions -- and to only pursue policies that have the support of the majority of House Republicans.
He has left the door open to working with the administration on border security, tax reform and highway funding.