Yau and Leung were disqualified by the city's High Court in mid November and lost an appeal to overturn that ban later in the month
Yau Wai-ching and Baggio Leung were elected in citywide polls in September but deliberately misread their oaths of office during their swearing-in ceremony, inserting expletives and draping themselves with "Hong Kong is not China" flags.
This prompted a rare interference by Chinese authorities.
The pair were amongst a handful of rebel candidates who took seats for the first time after the September polls, advocating either independence or self-determination for the southern Chinese city.
The new movement supporting a possible split from Beijing for the semi-autonomous city has gained traction as young pro-democracy campaigners grow increasingly frustrated with a lack of political reform.
Beijing took aim at the pair in a special "interpretation" of the city's constitution in early November that effectively barred them from taking up their seats because of the way they took the oath.
Following that decision, Yau and Leung were disqualified by the city's High Court in mid November and lost an appeal to overturn that ban later in the month.
On Wednesday the pair said they had filed appeal documents with the city's Court of Final Appeal, in hopes of setting a precedent for future elections in Hong Kong.
"It is shaping Hong Kong's constitutional environment, and even the legitimacy of Legislative Council elections hinge on this case," Yau told AFP.
The pair are still struggling to raise the funds to bring the case to the city's highest court.
"We're determined to appeal because we can’t stop at the Court of Appeal. The impact this case will have on Hong Kong is too big."
Following Yau and Leung's failed appeal last month, the government launched another legal bid to unseat an additional four pro-democracy lawmakers for failing to take their oaths properly, in what the opposition camp has called a witch hunt.
Proceedings against the four lawmakers are scheduled to start in February.
Thousands are expected to join a rally on New Year's Day calling for the government to drop their bid to disqualify the four lawmakers and to press for universal suffrage.