Background: Dengue infections are a severe public health problem in Brazil. The Ministry of Health recommends an immunosorbent assay (ELISA) for the capture of IgM (MAC-ELISA) to diagnose dengue. However, it detects antibodies that cross-react with other flaviviruses and requires confirmation in reference laboratories. Methods: One-step multiplex RT-PCR assay was used to amplify RNA of 197 serum from patients with clinical suspicion of dengue infection. The samples had been screened with the IgM ELISA kit in the Central Public Health Laboratory of the State of Maranhão. Results: Of the 197 samples evaluated by IgM ELISA, 135 were positive; of these, 96 (71.1%) were from patients in the acute phase of the infection. The one-step multiplex RT-PCR detected viral RNA in 88 (91.7%) of this serum. Among the 62-negative serum by ELISA, 29 samples (46.8%) were amplified using the molecular method. Conclusions: One-step multiplex RT-PCR was sensitive in the detection of viral particles from the first day until the sixth day after the onset of the feverish period. Moreover, it was specific and 100% reproducible. Based on these results, we recommend the use of this molecular assay to diagnose and differentiate the DENV serotypes in the acute phase of the disease.