The January 30 ruling, which cannot be appealed, comes as Prime Minister Andrej Babis has yet to form a coalition government since elections in October in which his party won 78 out of parliament's 200 seats.
Potential coalition partners are baulking over unanswered questions regarding Babis's communist past as well as the business dealings of the prime minister, who is the country's second richest person.
Lawyer Pavol Polacek, who represented the Slovak National Memory Institute in the case, said on Facebook: "The Bratislava court has made a full-stop to this lawsuit.
"There is no extant court verdict saying that Andrej Babis was registered as an StB agent illegally," he added.
The Slovak-born farm, food and media tycoon was a member of the Communist Party in the former Czechoslovakia in the 1980s, and his name appears in secret police files from that time.
Babis, 63, has denied any wilful cooperation with the StB, saying the police registered him without his knowledge when he worked for an export company in Czechoslovakia.
Babis's case came back to the regional court after Slovakia's constitutional court threw out previous court rulings in Babis's favour, arguing that the lower courts' verdicts had relied heavily on the testimony of former secret police agents.
After the elections, Babis managed only to form a minority cabinet largely made up of ANO members.
It lost a confidence vote and resigned in January, but has been allowed to continue to govern until a new cabinet is formed.
Babis said Tuesday he would keep suing to clear his name.
"Of course I'm not happy, I have never collaborated, I have never signed anything, and this is all made up," he told Czech Television.
Babis is also facing police charges over excluding a farm south of Prague from his sprawling Agrofert holding company in 2007 to make it eligible for an EU subsidy, before eventually taking it back.
Czechoslovakia split into the Czech Republic and Slovakia in 1993, four years after shedding four decades of totalitarian Communist rule.
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