If the court had ruled in EDF's favour, the firm would have been allowed to seek compensation -- but it would not have affected the deal, signed earlier this month by Prague and South Korea's KHNP.
The construction is crucial for the Czech Republic, which relies on nuclear power from two plants for 40 percent of its electricity consumption.
"The complaint has been rejected," Klara Belkovova, a spokeswoman for the Regional Court in the city of Brno, told AFP.
She said EDF had eight days to file an appeal with the Supreme Administrative Court to contest the verdict.
But EDF said in a statement obtained by AFP that it would "not pursue any further legal action in the Czech Republic", adding that it "takes note" of the Brno court's decision.
EDF has also filed a complaint over the tender with the European Commission, claiming KHNP may have used state aid that is illegal in the EU. The Commission has not yet decided on the case.
In the rejected complaint, EDF questioned the transparency of the bidding process.
But Prime Minister Petr Fiala said earlier that KHNP's bid was "better in all criteria assessed" than EDF's offer.
KHNP is due to build two nuclear reactors at the Dukovany plant for some 200 billion Czech koruna ($9 billion) each, with construction scheduled to begin in 2029 and the first reactor to be launched in trial operation in 2036.
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