The online and television advert, which was unveiled at the weekend by the Democratic Alliance, DA, has caused a stir on social media in recent days, with some South Africans calling the party "tone-deaf."
South Africa votes in parliamentary elections on May 29, in what is set to be the tightest vote since democratic rule was introduced at the end of apartheid.
Speaking to reporters during a visit to Limpopo province, Ramaphosa said the advert "is treasonous."
"It is the most despicable political act that anyone can embark upon," Ramaphosa said, accusing the DA of desecrating a symbol of national unity.
Zizi Kodwa, South Africa's Culture Minister said he would consider taking further steps "against the abhorrent and unpatriotic political advertisement."
The DA won the second largest share of the vote in South Africa's last elections.
The upcoming May 29 election is hotly contested, with polls suggesting the ANC will lose its majority for the first time since it came to power 30 years ago at the end of apartheid. This has raised the possibility that the governing party will have to form a coalition.
The advert shows a flag slowly burning as a voice warns of a disastrous alliance between the ANC and the Economic Freedom Fighters, EFF, South Africa’s fastest growing political party.
An intact flag then unrolls from the ashes of the burnt one as the slogan "Unite to rescue South Africa, vote DA" appears.
Helen Zille, the DA’s former leader and chairperson of the party's federal council wrote an opinion column where she said the opposition party "picked the flag as the clearest symbol of the dream we (South Africans) shared 30 years ago, at the dawn of democracy under President Nelson Mandela."
"The flames show how his vision has been ravaged by 30 years of ANC rule," she said.
Speaking on Radio 702, a local radio station, Zille said the blowback was coming from people who would never vote for the DA anyway.
"It creates a choice. Are you part of burning the flag? Are you part of that brigade or are you part of those of us like the DA who want to restore the flag?" Zille said.
John Steenhuisen, the DA's leader says a coalition government between the ANC and EFF would be a "doomsday" scenario for the nation. However, he did not rule out the prospects of partnering with the governing party in government.
"We can prevent our beautiful flag from burning, and we can prevent Doomsday — only if we come out in our millions to vote for the DA," Steenhuisen told a party conference.
Political tensions have mounted in the run-up to the polls between the DA and the ANC, which is struggling in opinion polls amid a weak economy and allegations of corruption and mismanagement.
The ANC, for its part, has said it will win the election and will not need to go into coalition. The latest opinion poll showed its support at just over 40%.
Information for this article was sourced from Reuters and Agence France-Presse.