Dengue virus universal and dengue serotype 1 to 4, fluorogenic probe hydrolysis (TaqMan), reverse transcription-polymerase chain reaction assays were developed for screening and serotype identification of infected mosquito vectors and human sera using a field-deployable, fluorometric thermocycler. Dengue universal and dengue 1 to 4 serotype assay in vitro sensitivity and specificity results were 100% concordant when tested with total nucleic acid extracts of multiple strains of dengue serotype 1 to 4, yellow fever, Japanese encephalitis, West Nile, and St. Louis encephalitis viruses. The in vitro sensitivity and specificity results for all five assays were concordant when tested with a blind panel of 27 dengue virus-infected mosquitoes, 21 non-dengue (yellow fever, West Nile, or St. Louis encephalitis) flavivirus-infected mosquitoes, and 11 uninfected mosquitoes and with clinical specimens consisting of a human serum panel of eight dengue viremic and 31 non-dengue-infected febrile patient serum samples. No cross-reaction occurred with vector species or human genomic DNA. Sample processing and polymerase chain reaction required <2 hours.