The court ruled that the ban, challenged by Corinne Cestino and Sophie Hasslauer, was in keeping with the constitution.
Activists had hoped France would join states like Spain and Belgium in legalising same-sex marriage.
An opinion poll suggests most French people are in favour.
The TNS Sofres survey of 950 people suggests that 58% of French people approve while 35% oppose gay marriage.
The idea that the court should rule at all on gay marriage was condemned by the leader of France’s far-right National Front, Marine Le Pen.
Fifteen years together
The court, or Constitutional Council as it is formally known, reached its decision through a panel of eight judges, six men and two women.
While many European states recognise homosexual civil unions, only Spain, the Netherlands, Belgium, Portugal, Sweden, Norway and Iceland legally acknowledge same-sex marriage.
Ms Cestino and Ms Hasslauer have lived together 15 years, are raising four children together, and already benefit from a French law recognising their partnership, but they cannot marry.
“It is not so much about getting married but about having the right to get married,” Ms Cestino, a paediatrician, told the Associated Press news agency.
“So, that is what we are asking for: just to be able, like anyone else, to choose to get married or not.”
At issue for the court was the legality of two articles in the civil code stipulating that marriage can only be between a man and a woman.
On a separate issue, that of whether gay couples were being discriminated against because the law treated them differently to heterosexual couples, the court ruled: “It is not up to the constitutional court to substitute its assessment for that of legislators.”
Lawyers for the lesbian couple are hoping the decision will encourage lawmakers to draw up a parliamentary bill on homosexual marriage, which could make the issue an election issue next year.
Under their civil union, the lesbian couple have tax benefits and other financial advantages.
But the couple told AFP news agency: “Marriage is the only solution in terms of protecting our children, sharing parental authority, settling inheritance problems and eventual custody if one of us were to die.”
After a Green Party mayor in the south-western town of Begles officiated over a wedding of two gay men in 2004, France’s highest court annulled the marriage.
Ms Le Pen said she was “totally” opposed to same-sex marriage and that the French people, not the constitutional court, should decide on its validity.
She said she believed that most homosexuals did not want the right to marry either.
“The vast majority of homosexuals are not demanding the right to be different but the right to be left alone,” the far-right leader said. – Bbcnews