The House voted 311-114 to immediately remove the controversial freshman congressmen, above the two-thirds majority required to oust one of its own.
Shortly before the vote, House Speaker Mike Johnson said he would vote against the expulsion, an aide said.
Santos, 35, has been mired in controversy since his November 2022 election. He has admitted fabricating much of his biography, and federal prosecutors accuse him of laundering campaign funds and defrauding donors. Santos has pleaded not guilty to those charges.
He survived a previous expulsion attempt in early November, when 182 of his fellow Republicans and 31 Democrats voted against his removal on the grounds that his criminal case should be resolved first. His expulsion reduces Republicans' already slim majority to a 221-213 majority. His district, which includes parts of New York City and Long Island, is seen as competitive.
A bipartisan congressional investigation last month found that he charged almost $US4000 ($A6,045) for spa treatments, including Botox, to his congressional campaign account. He also spent more than $US4000 ($A6,045) of campaign money at the luxury retail store Hermes and made "smaller purchases" from OnlyFans, an online platform known for sexual content.
That prompted several Republicans who backed him in November's vote to say they would support kicking him out. Santos has said he likely would be expelled.
He spurned calls to resign but had said he would not run for reelection next year.
Santos is only the sixth member to be expelled from the House, and the first who has not been convicted of a crime or fought for the Confederacy during the 1861-65 US Civil War. Democrat James Traficant was the last member to be expelled in 2002, following his criminal corruption conviction.
On the House floor on Thursday, Santos said: "I have been convicted of no crimes. The people of Third (Congressional) District of New York sent me here.
"If they want me out, they're going to have to go silence those people and take the hard vote."
Santos' troubles began shortly after his November 2022 election, when media outlets reported he had not actually attended New York University or worked at Goldman Sachs and Citigroup, as he had claimed during the campaign.
He also falsely claimed Jewish heritage and told voters his grandparents had fled the Nazis during World War Two.
"Every day that Mr Santos is allowed to remain a member of Congress, my New York neighbours to the west are being denied real representation in these halls," Representative Nick LaLota, a Republican representing a neighbouring district in New York, said on the House floor on Thursday.
Reports of the falsehoods made Santos a pariah in the House and the butt of late-night TV comedians even before federal prosecutors charged him with an array of fraud and campaign-finance crimes.
In a 23-count indictment, they accuse him of inflating his fundraising totals in order to draw more support from the Republican Party, laundering funds to pay for personal expenses, and charging donors' credits cards without permission.
Two former campaign aides have pleaded guilty to related fraud charges.
Santos denies wrongdoing, and his trial is scheduled to begin on September 9, 2024, shortly before the November elections that will determine control of the White House and both chambers of Congress.
