Insect-specific flaviviruses (ISFs) are a group of orthoflaviviruses that can replicate efficiently in arthropods but are unable to replicate in vertebrate hosts. This contrasts with medically important orthoflaviviruses, such as dengue virus (DENV) and West Nile virus (WNV). Using the established monoclonal antibody against viral RNA intermediates in cell assay, we report the first isolation of an ISF, Quang Binh virus (QBV), in Singapore from three pools of Culex gelidus mosquitoes. To determine the spatiotemporal prevalence of QBV, a total of 17,070 mosquitoes, represented as 721 pools, were screened using a QBV-specific reverse transcriptase quantitative PCR assay, revealing 36 QBV-positive pools of mosquitoes detected mainly along the northern coastal regions of Singapore. Repeated detections over 12 months in a north-western nature reserve suggest local stable establishment of the virus. Phylogenetic and molecular analyses show that QBV sequences from the Singapore group together with other Southeast Asian sequences and that Cx. gelidus-derived sequences are phylogenetically distinct from those derived from Culex tritaeniorhynchus. We also present in vitro evidence that QBV is able to suppress DENV2 and WNVKUN in C6/36 cells by 2.9 logs and 1.8 logs, respectively. This report represents the first known spatiotemporal study of an ISF and highlights QBV’s potential as a biological control against medically important orthoflaviviruses.