Luisa Ortega, the highest-ranking public official to openly defy Maduro in the crisis, told reporters she had filed a case with the constitutional court on human rights grounds.
Her move is a challenge to a court ruling a day earlier which held that Maduro could set up an elected constitutional reform body without holding a referendum to approve it.
Opponents of the socialist president say he aims to keep himself in power by stacking the planned "constituent assembly" with his allies.
Ortega has been a traditional ally of the socialist leadership, but her criticism of Maduro over the past two months of violent unrest have raised the prospect of divisions in the government camp.
Elected in 2013, Maduro is resisting opposition calls for early elections to remove him.
The opposition blames him for severe food and medicine shortages. He says the crisis is a US-backed conspiracy.
Talks between the government and opposition broke down in December with the sides accusing each other of bad faith.
Maduro's Foreign Minister Delcy Rodriguez told a news conference Thursday the government was inviting five Latin American and Caribbean countries to act as mediators in a new round of talks.
The opposition had previously ruled out a return to such negotiations.
Anti-government protests and looting have been raging across Venezuela for the past two months.
Riot police have fired tear gas and plastic bullets against protesters, who have hurled rocks and Molotov cocktails.
State Ombudsman Tarek William Saab at a news conference on Thursday called on the security forces to "fulfill their mission to protect human rights" in their response to the protests.
He said 65 people have been killed over two months of unrest. The state prosecution service puts the toll at 60.
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