The plate centrifugation assay was standardized for dengue virus isolation from serum samples. C6/36-HT cells were used determining the optimal values for centrifugation spin speed, inoculum, sera dilution, and incubation time. Then, 22 positive serum samples with viral isolation and viral strains of the four reference dengue virus serotypes were tested simultaneously by the standardized plate centrifugation method and the conventional tube culture. The isolations were typified by indirect immunofluorescent test using monoclonal antibodies. The plate centrifugation method was optimized to 200 μL of inoculum, dilution of sera 1/20, centrifugation speed at 1600 rpm/30 min, and sensitivity of 95,5% after 5 days post-inoculation. We concluded that the plate centrifugation method increased dengue virus isolation, with a significant reduction of the time of isolation for dengue virus.