Alexandre Cloutier, the Parti Quebecois' minister of intergovernmental affairs, called the move a "direct" and "devious" attack against the Quebec nation.
Federal NDP Leader Tom Mulcair, meanwhile, suggested the move is an unnecessary provocation that plays into the separatist government's hands.
However, Harper got plaudits from Liberal intergovernmental affairs critic Stephane Dion, the architect of the federal law on secession which first prompted Quebec to adopt its own, contrary rules for separating from Canada.
Cloutier said only Quebec and its own legislature, the national assembly, have the right to determine whether the province stays in Canada — not the federal government.
"Obviously, they want to make sure we'll never get our country," Cloutier said Sunday at a news conference in Quebec City.
A statement from federal intergovernmental affairs minister Denis Lebel dismissed Cloutier's comments saying the Harper government has no intention of re-opening the constitutional debate.
"Bear in mind that the federal government did not initiate the proceedings," Lebel said in the statement.
"We are involved because it is a question of Canadian unity. It is completely normal for the federal government to defend Canadian laws."
Bill 99 declares that a referendum vote that gets 50 per cent plus one in favour of independence should be considered enough to separate from Canada.
The federal government finally filed a legal intervention against the law last week, entering into a court challenge launched by a Quebec English-rights activist shortly after it was first adopted.
The challenge, filed 13 years ago by Keith Henderson, argued Bill 99 is invalid under the Canadian Constitution.
For years, the federal government's strategy appears to have been to delay the court case. But with a trial date to be set this December in Quebec superior court, Ottawa decided to enter the fray.
The federal government argues in its intervention that Bill 99 does not provide the legal basis for a "unilateral declaration of independence by the government."
Mulcair said the federal intervention is an unwelcome and unnecessary revival of constitutional debates that have remained mostly dormant for more than a decade.
"It's for nostalgics to go back to this," Mulcair said in an interview, noting the court case has been "floating around in the miasma" for 13 years.
"There's no question that this is a life buoy, a life saver for the sovereigntists who see this as a great way to start an old battle with Ottawa and there's not much to be gained by it."
While support for independence is low in Quebec, the PQ, which holds a minority in the legislature, could use a battle with Ottawa to drum up support in an election. An election could be called as early as next month.
Bill 99 was introduced in the year 2000 by Lucien Bouchard's PQ government in response to the federal Clarity Act, which states that clear majority support on a clear referendum question would be required to trigger negotiations on secession.
While Bill 99 did not, in the final vote, receive the support of Jean Charest's Liberals, they expressed agreement with some of its fundamental principles. Mulcair was part of Charest's caucus at the time.
Mulcair suggested Sunday that Bill 99 is effectively meaningless since it can't override the Supreme Court of Canada's ruling on secession.
The top court ruled in 1998 that Quebec could not legally secede unilaterally. However, the rest of the country would be obligated to negotiate a constitutional amendment on secession if a clear majority of Quebecers were to vote in favour of a divorce, the court said.
Dion, who crafted the Clarity Act based on the top court's advice, said the Harper government had to intervene in the Bill 99 court fight "because it is its duty to protect the rights of Quebecers" to a clear legal process for determining whether the province remains part of Canada.
He said the PQ is deliberately trying to confuse the issue, arguing publicly that Quebec has the right to unilaterally secede while arguing in court that Bill 99 is simply a statement of principles.
"This double talk should be denounced by everyone," Dion said.
Describing the law "one of the most important in Quebec history," Cloutier said he will instruct government lawyers to do everything they can to fight the federal court challenge.
He added the PQ will introduce a motion next week in support of Bill 99 and expects the support of all political parties.
But while Cloutier came out forcefully on Sunday, his government's own lawyers have taken a far different approach in addressing the court challenge.
In fact, a defence filed in the case recently by the Quebec government argues Bill 99 doesn't conflict with the Canadian constitution. Instead, it contends that Bill 99 reaffirms the "fundamental rights" of Quebecers that are consistent with Canadian law.
While Mulcair slammed Harper for reviving the debate and mocked the federal Liberals for sending "the hand of Stephane Dion out of the crypt" to inflame it further, he said the NDP is moving forward with a more positive approach.
The NDP contends that a 50 per cent plus one referendum vote would be sufficient to trigger negotiations on secession, provided the referendum question was clear and there were no irregularities in the vote. Mulcair insisted that's consistent with the Supreme Court's ruling.
Also on HuffPost