Background: Virological surveillance provides an early warning sign for the risk of transmission in an area. Laboratory tests for dengue virus infection on mosquitoes include isolation of the virus, Polymerase Chain Reaction (PCR) and Direct Fluorescent-Antibody (DFA) requires a high level of technical skill, expensive equipment, and time-consuming. A method based on immunocytochemical (IC) using monoclonal antibody DSSE10 has several advantages. This study aimed to evaluate sensitivity and specificity IC assay compared with Reverse Transcription-Polymerase Chain Reaction (RT-PCR) as gold standard to detect Dengue Virus (DENV)-3 infections in mosquito Aedes aegypti.Methods: An experimental study was conducted in laboratory of Medical Parasitology, Faculty of Medicine, Universitas Gadjah Mada (UGM) in May 2009 until October 2010. A total of 22 artificially-infected adult Ae. aegypti mosquitoes of DENV 3 were used as infectious samples and 35 non-infected adult Ae. aegypti mosquitoes were used as normal ones. The IC Streptavidin Biotin Peroxidase Complex (SBPC) assay using monoclonal antibody DSSE10 was applied in mosquito head squash to detect Dengue virus antigen. RT-PCR as a gold standard was applied in mosquito thorax.Results: The kappa value showed a good agreement between two observers (kappa value 0.63). IC could detect dengue virus antigen as sensitive as RT-PCR (sensitivity 100%). But IC was less specific than RT-PCR (specificity 91%) because some false positive results were found in this method.Conclusion: The IC method has a high sensitivity and high specificity compared with RT-PCR. This IC method may be useful for virological surveillance of dengue infected Aedes mosquitoes. (Health Science Indones 2011;2:87-91).