Internet providers appealed the ruling from Copenhagen City Court, as they claimed that a website could only be blocked if the regulator could prove that Danish customers had actually played on the site in question.
However, the decision was upheld by the High Court, meaning the case is now closed and the 55 web pages remain blocked.
Spillemyndigheden initially ordered the websites in question to halt operations, but took the case to court after they failed to do so.
During the hearing on 2 March, the regulator was able to prove that all of the sites had been offering games in Denmark without a licence and were therefore in breach of national regulations.
The sites were divided into five categories by the regulator; online casino; online casino and lottery; online casino and online betting; online casino, lottery and online betting; and skin betting.
Among the sites blocked were Easybet.com, Slotjoint.com and KingBillyCasino.com. Meanwhile, 23 of the sites were in the esports skin betting category.
The case represents the highest single incidence of website blocking by the regulator since it began blocking websites in 2012.