Yau Wai-ching and Baggio Leung had won seats in citywide polls last September, but were ousted from Hong Kong's legislative council for taking improper oaths.
They belong to a new movement calling for a complete split from China for semi-autonomous Hong Kong, as fears about Beijing's tightening grip on the city grow.
At that ceremony, they draped themselves in "Hong Kong is not China" flags and altered the wording of their pledges, including derogatory terms and expletives.
Hong Kong's High Court in November ruled the oaths of the two pro-independence lawmakers were invalid, in an unprecedented judicial review brought by the city's leader and justice secretary.
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That ruling came after Beijing issued a special interpretation of the city's constitution to insist oaths be taken in a "sincere and solemn" manner.
The city's Court of Final Appeal on Friday dismissed the duo's applications to appeal, ending their bid to overturn the ruling that ousted them from the legislature.
During the two-hour hearing, David Pannick, the lawyer representing Leung said that the oath taking should be an internal matter for the city's legislature.
Following the decision, Leung said he and Yau were "extremely disappointed".
"If I had known of the results, I will not have run in this election," Leung told reporters outside the courthouse.
"When a process such as an election which empowers the people can be affected by a government that was not authorised by the people, I don't know why I would run in such an election," he said.