The proposed Employment Non-Discrimination Act "will increase frivolous litigation and cost American jobs, especially small-business jobs," the Ohio Republican said through a spokesman.
Boehner has said the law is unnecessary because gays and lesbians are covered under existing law.
"We have always believed this is covered by existing law," a Boehner aide told The Huffington Post.
The speaker's opposition is "not a new issue or a new position -- it's a longstanding position, and, frankly, not 'news' at all," the aide said. "This has been his position, on the record, for years, stated publicly many times."
Federal law protects people from workplace discrimination because of race, religion and other factors.
The 2009 Matthew Shepard and James Byrd Jr. Hate Crimes Prevention Act, also known as the Matthew Shepard Act, expands U.S. hate-crime law to include crimes motivated by a victim's actual or perceived gender, sexual orientation, gender identity or disability.
But it's legal in most states to fire or refuse to hire people because they are gay, lesbian, bisexual or transgender.
Just 21 states and the District of Columbia offer protections against such discrimination to varying degrees.
ENDA, as proposed, would prohibit discrimination in hiring and employment on the basis of sexual orientation or gender identity by civilian, non-religious employers with at least 15 employees.
Other House Republicans have also said they would oppose the bill, which passed the Senate 61-30 Monday night and moved to the Senate floor after seven Republicans crossed party lines and voted to begin debate on it.
Supporters of the bill -- introduced in every Congress except one since 1994 -- say their best chance for House passage is to try to attach it to an unrelated measure authorizing defense programs, The Wall Street Journal reported.
Tuesday's debate will be the first time since 1996 the full Senate will consider a measure to extend federal non-discrimination law to gay, lesbian and bisexual people.
Senators are widely expected to try to amend the bill to address Republican concerns.
One proposed change would expand the types of religious groups exempt from the bill, The New York Times said. Another would guarantee religious institutions are not subject to federal retaliation if they refuse to employ people covered under the bill.
The Washington Post said Monday night's vote all but guaranteed full Senate passage as soon as this week.
"Here we are today, now taking one more step to make the American family more inclusive," said Sen. Tom Harkin, D-Iowa.
"No one should be discriminated against because of race, sex, religion, national origin, and they shouldn't be discriminated against because of who they love or who they are," he said.
Sen. Susan Collins, R-Maine, said many of her constituents were shocked when they learned federal non-discrimination protections don't already exist.
"They just assume that our civil rights laws, which protect people from discrimination based on race, gender, religion, age, also protect individuals based on sexual orientation," she said.
Collins called the measure "common sense."
Sen. Mark Kirk, R-Ill., used his first Senate floor speech since suffering a stroke two years ago to support the bill.
Sen. Lisa Murkowski, R-Alaska, who voted for the legislation in committee and supports same-sex marriage, was absent.