Abstract: Objective：To establish a double fluorescent quantitative RT-PCR assay for simultaneously detecting coxsackievirus A2 (CVA2) and A5 (CVA5) subtypes. Methods：The specific primers and probes of CVA2 and CVA5 subtypes for fluorescent quantitative RT-PCR assay were designed with Primer Express 3.0 software. The optimizing double fluorescent quantitative RT-PCR system with a standard curve was established, and its specificity, sensitivity and repeatability were evaluated. Then, the stool specimens from 367 children suspected with hand-foot-and-mouth disease were detected by this assay, and the positive specimens were further sequenced. Results：The established double fluorescent quantitative RT-PCR was able to detect CVA2 and CVA5 subtypes specifically, and had no cross reaction with other enteric viruses. Its lowest detectable limit was 10² copies /mL, and the intra assay coefficients of variation were all less than 1.49%. The positive rates of CVA2 and CVA5 subtypes in 367 clinical specimens were 6.3% (23/367) and 3% (11/367), respectively, and the positive results were verified by subsequent sequencing. Conclusion：The double fluorescent quantitative RT-PCR assay for simultaneously detecting CVA2 and CVA5 subtypes in a single test tube is successfully established, which has good specificity, sensitivity and repeatability, and may be used to rapidly screen the infections caused by CVA2 and CVA5 subtypes and investigate the epidemiology of hand-foot-and-mouth disease.