The legislation was one of the two triggers for the July double dissolution that sent the country to an election.
The second trigger, to re-establish the Australian Building and Construction Commission, is still being negotiated with the crossbench.
The vote on the bill came after another Senate vote last night where National Party senators voted against the Government over a ban on the Adler shotgun.
It was just after 2am on Tuesday when theTurnbull Government's Registered Organisations Bill passed through the Senate.
The bill, which deals with union corruption, was one of two key pieces of legislation used to trigger July's double-dissolution election.
Crossbenchers Derryn Hinch and Nick Xenophon backed the bill after the Government agreed to amendments to protect whistleblowers.
Senator Xenophon says they have secured what could be the best whistleblower protections in the world.
"These are momentous changes to whistleblower-protection laws that the Government has committed will extend to the corporate and public sectors. They will include, for the first time, a broadening of the definitions of what a reprisal action is, mechanisms to make sure what the level of harm is to individuals much broader than the current public-interest-disclosure legislation across Australia, which has proved to be woefully inadequate in respect to this."
Senator Hinch argued the changes were a necessary safeguard for whistleblowers.
"It would cover anonymity, compensation and protection. And even though it now deals specifically with unions, it must, in the near future, be extended with the same powers and the same protection to whistleblowers in the corporate section."
Labor, the Greens and Tasmanian senator Jackie Lambie opposed the bill.
Senator Lambie questioned the Government's decision to compromise with the crossbenchers, claiming it was not willing to do so before the election.
"I would like to know why we all went to a double-D (dissolution) and you weren't prepared to compromise."
The Government has argued the bill is needed to deal with widespread corruption uncovered by the Trade Union Royal Commission.
The bill creates a new specialist regulator, the Registered Organisations Commission, that takes over responsibility from the Fair Work Commission to oversee registered unions and employer associations.
The Government's deputy leader in the Senate, Mathias Cormann, says passage of the bill means unions will be regulated in the same way as company directors.
"It was a very significant achievement in the national interest, ensuring that union officials and other officials in registered organisations now have to comply with similar transparency and accountability standards as company directors already do. And this will, of course, better protect hard-working union members from being ripped off by bad union officials."
The move came after another Senate vote earlier in the night on the importation of the Adler firearm, where two Nationals senators voted against the Government.
Liberal Democrat senator David Leyonhjelm had put forward a motion to lift a ban on the importation of the shotgun.
But the major parties and the Greens voted against it, despite Nationals senators Bridget McKenzie and John Williams crossing the floor to vote with Senator Leyonhjelm.
Nationals ministers Nigel Scullion, Fiona Nash and Matt Canavan were not in the chamber for the vote.
But Senator Williams has told Sky News his colleagues were aware of how he would vote.
"When I've spoken to some of my team today, my leader Nigel Scullion -- and he was well-aware of it -- Fiona Nash, deputy leader of the party, we just discussed the issue, and we just thought we needed to stand up for the innocent, honest, gun owners here."
But the leader of the Opposition in the Senate, Penny Wong, says it is not a good look for the Government.
"I don't know if others know this, but the Nationals ... the Nationals voted against the Government. The Turnbull Government's split on the floor of the Senate, the Nationals voting with Senator Leyonhjelm. And where were the National ministers? Where were the National Cabinet ministers?"
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