Movie director Kim Jho Gwang-soo and his long-time partner Kim Seung-hwan are the most high-profile gay couple in South Korea.
The pair held a wedding ceremony in Seoul in September 2013 and submitted their marriage registration form to their local authority, but it was rejected.
In July last year, the couple challenged the decision with a lawsuit—the first of its kind in the country.
However, on Wednesday the Seoul western district court sided with the local authority and rejected the challenge.
"Circumstances have changed concerning marriage ... but unless there is separate legislation, a same sex union cannot be recognised as marriage under the existing legal system", the court said in a statement.
"Related laws, including the constitution and civil law, are premised on the notion of a conjugal bond meaning a union involving different sexes," it said.
The couple's lawyer, Ryu Min-Hee, admitted that the likelihood of a district court ruling in their favour was very low and said "We're disappointed but we're not done yet," according to AFP.
"We regret the court decision ... but we did see it coming," Kim Seung-Hwan said. The couple intended to appeal the ruling.
Homosexuality is legal in South Korea but the the country remains deeply conservative and same-sex marriage is not recognised.