Lecuona missed last weekend's European Grand Prix at the same circuit after having to quarantine for 10 days because his brother, with whom he lives in Andorra, had tested positive.
Andorran authorities cleared him to travel to Valencia after he tested negative on Wednesday, but the 20-year-old returned a positive test on Saturday morning.
KTM Tech3 said Lecuona was not showing any symptoms.
"We have sad news today. Although Iker has been tested negative three times, the last test he took this morning at the entrance of the circuit showed positive," team manager Herve Poncharal said in a statement.
"Therefore, he won't be able to enter the circuit and participate at the Gran Premio de la Comunitat Valenciana.
"He will be tested furthermore in order to understand if ever he can fly to Portugal to participate at the last Grand Prix of the season in Portimao."
Lecuona is 17th in the standings with 27 points. He is the second MotoGP rider after Italian Valentino Rossi to miss a race this season due to the novel coronavirus. (Reporting by Shrivathsa Sridhar in Bengaluru; Editing by Kim Coghill)