WADA had expressed its disappointment with the judge's decision last month to order evidence including bags of blood to be destroyed, depriving the agency and other sports bodies of the chance to analyse them.
The Spanish doctor at the centre of the trial, Eufemiano Fuentes, had told the court that as well as cyclists his clients included athletes in sports such as soccer, athletics, tennis and boxing.
"The World Anti-Doping Agency (WADA) is filing a declaration to appeal to the Criminal Court in Madrid in relation with the Operation Puerto," WADA said in a brief statement on their website (www.wada-ama.org).
"Following its policy, WADA will refrain from commenting further in order to protect the integrity of the process," the statement added.
WADA's appeal joins those already filed by the Spanish prosecutor and the Italian Olympic Committee (CONI).
Other bodies including Spain's anti-doping agency (AEA) have said they will challenge the judge's ruling and have until the beginning of next month to file their appeals.
The judge's decision not to release the evidence for further investigation dented hopes that the case would unmask other athletes involved in doping and prompted widespread condemnation.
Spain hoped the trial would help dispel the impression that the nation was soft on doping and boost Madrid's bid to win the right to host the 2020 Olympic Games.
The country is pushing updated anti-doping legislation through parliament which the government says will bring Spain into line with international norms. (Reporting by Iain Rogers, editing by Toby Davis)