The aim of this study was to retrospectively determine the prevalence of dengue virus from April 2010 to November 2010 in Brunei Darussalam. A total of 250 serum samples from dengue diagnosed patients were examined. All serum samples were tested for dengue IgM and IgG antibodies and dengue NS1 antigen using the PanBio dengue ELISA commercial kit. To determine the prevalence of dengue virus serotype in the country, serotyping was performed for the 14 samples that were positive for NS1 antigen. Dengue virus serotyping was carried out using the conventional reverse transcriptase-polymerase chain reaction (RT-PCR). Of the 250 serum samples included in the study, 196 were laboratory dengue confirmed cases. Dengue virus serotype 1 (DENV-1) was the predominant circulating serotype, followed by DENV-3 and DENV-2. This is the first report of DENV-3 isolation in Brunei Darussalam.