The announcement by the widely-respected Makoni comes after the breakdown of talks between the two factions of the main opposition Movement for Democratic Change designed to agree on a joint candidate to take on 83-year-old Mugabe.
A spokesman for the faction of the opposition Movement for Democratic Change led by Arthur Mutambara welcomed Makoni's announcement.
"We welcome anyone who is willing to fight poverty, hunger and the dictatorship of Robert Mugabe's government," Gabriel Chaibva said.
Takavafira Zhou, an analyst at Zimbabwe's Masvingo State University, said Makoni would present a stiff challenge, especially if he could attract other disillusioned members of the establishment.
"The move by Makoni could mean the demise of Mugabe particularly if Makoni can get the support of the Mujurus and the army," he said.
Joyce Mujuru, the vice president, and her husband Solomon Mujuru, a former head of the armed forces, have previously been suggested as possible challengers but have been reluctant to break ranks.
The 57-year-old Makoni said he had hoped to have stood as Zanu-PF's official candidate but had been left with no alternative after the party congress in December endorsed Mugabe to run for a sixth term.
George Katito, a Zimbabwe analyst at the Johannesburg-based South African Institute of International Affairs, said Makoni would face formidable hurdles.
"His challenge will breathe new life [into the contest] but whether it's a feasible platform is questionable considering the very strong political machine that Mugabe has run," he said.