**Abstract**

Anthropogenic actions, including deforestation, disorganized urbanization, and globalization, contribute to emergence and reemergence of arboviruses worldwide, where Flavivirus is the most prevalent, and its continuous monitoring can help in preventive control strategies. Thus, the aim of this study was to detect flavivirus RNA in single hematophagous insects, which are used as sentinels. Total RNA was extracted from six *Aedes aegypti* stored since 2003 and from 100 Culicidae and collected through CDC trap in a public park of a Brazilian Northwest city of São Paulo State. *Flavivirus* was detected through RT/PCR targeting 230–250 bp of the RNA polymerase coding sequence (NS5). PCR amplicons were sequenced by Sanger method, used in comparative analysis over Basic Local Alignment Search Tool (BLAST) in GenBank, and subjected to Neighbor-Joining phylogenetic analyses. Efficiency of *Flavivirus* diagnosis was confirmed by detection of Dengue virus serotype 2 in *Ae. aegypti*. From the 100 collected insects, 19 were positive for *Culex flavivirus* (CxFV). NS5 partial sequence phylogenetic analysis clustered all CxFV in one branch separated from vertebrate flaviviruses, being applicable to the identification of *Flavivirus* species. The dipteran RNA extraction methodology described in this work supports detection of flaviviruses in single insects maintained in 80% ethanol, which can be used to constant arbovirus surveillance.

**Keywords:** dipteran, *Flaviviruses*, hematophagous insect, molecular diagnosis, RNA extraction, single-insect virus diagnosis
