This will give the main opposition party a respected black figurehead to challenge the ruling ANC.
Mamphele, a medical doctor and former World Bank managing director, commands wide admiration among South Africa's black majority as the partner of Black Consciousness leader Steve Biko, beaten to death in apartheid police custody in 1977.
However, her Agang political party launched a year ago has struggled to gain traction despite growing disaffection among voters with the performance of the African National Congress (ANC), which has run the continent's biggest economy since the end of white-minority rule in 1994.
"I can think of no better person to be our presidential candidate in this crucial election," Democratic Alliance (DA) leader Helen Zille told a news conference, before embracing her new political ally.
Ramphele - also a successful businesswoman who made millions as a senior mining industry executive - alluded to the death in December of Nelson Mandela, South Africa's first black president, as a symbol of the changing political landscape.
"I believe this decision is in the best interests of South Africa as we head into turbulent waters," she said. "The death of Nelson Mandela has changed many things for South Africa."
Mamphele's inclusion as a top DA candidate is likely to help the party tackle the perception that it is the political home of privileged whites, but it is unlikely to have a major effect on the outcome of the presidential and parliamentary elections expected in May.
The ANC won nearly two thirds of the vote in the last elections in 2009 and even though its support has been waning since the advent of multi-racial democracy, its overall majority is not in question.