"The yeas are 60, the nays are 39. H.R. 3590 as amended, the Patient Protection and Affordable Care Act is passed," Vice President Joe Biden announced.
The vice president presided over the Senate at the time of the vote.
As expected, Republicans voted against the bill while all Democrats and two Independents voted for it.
At an estimated $87 billion, the measure would expand health insurance coverage to about 30 million more Americans currently without it, create new private insurance marketplaces, or exchanges, to expand choice.
And, like the slightly more expensive measure passed by the House of Representatives, it would end a practice by private insurance companies of denying coverage to individuals with existing health problems.
Both the Senate and House measures would require nearly all Americans to purchase some form of insurance, while lower-income Americans would receive help from federal government subsidies.
In remarks before the vote, Senate Democratic leader Harry Reid said opponents had done everything they could to prevent the vote from taking place.
Speaking to reporters, Reid and others hailed the vote as a victory and a major step toward providing millions more Americans with access to health care.
"This is a victory because we have affirmed that the ability to live a healthy life in our great country is a right and not merely a privilege for the select few," Reid said.
Reid and others paid tribute to to Senator Edward Kennedy, who died this past August after spending decades of his career in the Senate pursuing health care reform.
In the final hours of debate on the Senate bill, Republicans asserted it would be ineffective and add sharply to the U.S. budget deficit. Jeff Sessions is a Republican from Alabama:
"This legislation may have a great vision, it may have a great idea about trying to make the system work better. But it does not. These are huge costs [and] its not financially sound," Sessions said.
Ahead are difficult negotiations with the House of Representatives to craft a final bill President Obama would sign into law.
These talks, which will formally get under way early in the new year, will take place amid anger among many liberal House Democrats the Senate bill failed to contain a government-run insurance option.
Members of the House Progressive Caucus have vowed to fight to keep this public option in any final legislation that emerges, along with other provisions they say are needed to protect lower and middle-income Americans and hold insurance companies accountable.
In a statement, the Democratic chairmen of three key House committees said while there are clear differences between House and Senate bills, both will bring fundamental health care coverage to millions who are currently uninsured.
Obama administration officials have been quoted as saying they anticipate negotiations on a final bill would not be complete until after the president's State of the Union Address in January, and could slip even later into the new year.
US Senate Approves Historic Health Care Reform Bill