Republicans are expected to again block the bipartisan measure from advancing.
For the second time in three months, an immigration and border security measure negotiated earlier this year by a bipartisan group of lawmakers failed to advance in the Senate, drawing the issue to a political stalemate.
The legislation fell well short of the 60 votes needed -- the final tally was 43 to 50 -- as nearly all Republicans voted against it.
The showdown vote came as both parties try to establish themselves as tough on border security ahead of the 2024 election.
Majority Leader Chuck Schumer announced over the weekend plans to bring the legislation back up for a stand-alone vote, after it had earlier been tied to aid to Ukraine and Israel.
Schumer said ahead of Thursday's vote that the bill presented his colleagues on both sides of the aisle with a chance to demonstrate whether they're serious about fixing the challenges on the southern border, though he pointedly criticized Republicans for previously blocking the legislation at the direction of former President Donald Trump.
"If Republicans were truly serious about calling the situation at the border an emergency, they shouldn't delay any longer. You can't call something an emergency one day and then suddenly kick the can down the road the next day," Schumer said in floor remarks.
On the other side of the aisle, Senate Minority Leader Mitch McConnell, the top Republican in the chamber, criticized Democrats for "suddenly chomping at the bit" to pass a comprehensive border security bill and said their renewed efforts amounted to "cynical Senate theater."
Other Republicans, including one who helped draft it, also alleged the vote was a purely political effort by Democrats to distract from Biden's record on the border.
"The bill is no longer a bill and now it's just a prop," Sen. James Lankford, R-Okla., who co-wrote the bill alongside Connecticut Democratic Sen. Chris Murphy and Arizona Independent Sen. Kyrsten Sinema. "And that's been my frustration. We wrote it to be a bill to try to actually solve the problem."
Serious discussions about a bipartisan border security package first began in the late fall of 2023 after Senate Republicans, looking to capitalize on interest in securing additional foreign aid to Ukraine, said they would not support advancing a foreign aid package unless Congress passed serious legislation to regulate the Southern border.
Murphy, Sinema and Lankford huddled behind the scenes for months before ultimately unveiling their $20 billion proposal for the border that increased immigration restrictions and enforcement and implemented new migrant policy.
That package was originally meant to be tied to a broader national security supplemental that included aid to foreign allies Ukraine, Israel and Taiwan in an effort to secure the necessary Republican votes to pass foreign aid.
Though bipartisan talks were initially championed by Republicans, things began to falter after Trump, flexing his grip on the party ahead of the 2024 election, urged Republicans to block the legislation if it was anything less than perfect.
During a campaign rally in January, Trump told senators to "blame it on me" if the bill failed. Republicans quickly fell in line with Trump, and by the time the bipartisan group unveiled their product, its fate was all but sealed.
During the first vote to try to advance the legislation back in February, all but four Republicans voted against moving forward with the combined border and foreign aid package.
Democrats fiercely accused Republicans of cow tailing to their presumptive presidential nominee who they alleged wanted to preserve the crisis at the border to use as a campaign issue. Republicans, meanwhile, said the bill did not go nearly far enough to address the border crisis.
The Senate did eventually rubber stamp billions in foreign aid to Ukraine and other foreign allies in the weeks that followed, but the painstakingly negotiated bipartisan border provisions languished.
That was until Thursday, though the bill lost support on both sides in the latest round of voting.
Two of the bill's three authors, Sens. Lankford and Sinema, were among those who previously supported the legislation who voted against it on Thursday.
And despite the push from Democratic leadership, the bill was rejected by even some in the Senate Democratic caucus, especially by some progressives. Democratic Sens. Cory Booker and Laphonza Butler flipped their votes to no.
Schumer's move to once again attempt passage of a border bill came in late May of an election year when immigration and border security are top issue for voters on both sides of the aisle.
The fraught political environment had done little to warm bipartisan negotiations over the border, and in both the lead up and fallout from Thursday's vote, each party has accused the other of playing politics.
The White House had been in communication with all four Congressional leaders, including McConnell and House Speaker Mike Johnson, ahead of the vote.
President Joe Biden on Thursday, after the vote failed, criticized Republicans for "partisan politics ahead of our country's national security" and said he will "not stop fighting to deliver the resources that border and immigration personnel need."
"Congressional Republicans do not care about securing the border or fixing America's broken immigration system," Biden said. "If they did, they would have voted for the toughest border enforcement in history. Instead, today, they put partisan politics ahead of our country's national security.
Speaker Johnson, in a statement after the measure failed to advance, accused Schumer of "wasting time" on a bill he said would never have a chance of passing the House.
"After more than three years of claiming the situation at our southern border was not a crisis while millions of illegals poured in, Congressional Democrats are attempting to throw an election year Hail Mary to cover for their embrace of President Biden's open border policies," Johnson said as he again called on Biden to take more aggressive executive action on the border.
ABC News' Arthur Jones II and Mariam Khan contributed to this report.