Hand, foot and mouth disease (HFMD) is a serious public health problem. Generally, it is considered that HFMD is mainly caused by enterovirus 71 (EV71) and coxsackievirus A16 (CVA16). Nevertheless, the incidence of HFMD caused by coxsackievirus A6 (CVA6) and coxsackievirus A10 (CVA10) has increased significantly and CVA6 and CVA10 have become major causes of HFMD epidemic. This study develops a duplex real-time reverse transcript polymerase chain reaction (RT-PCR) assay for simultaneous detection of CVA 6 and CVA 10. The specificity, sensitivity, and reproducibility of this assay were analyzed. No cross-reactions with other viruses or false positives were observed. The detection limit of this assay was as low as 11.935 copies for CVA6 and 17.591 copies for CVA10 per reaction (concentration giving a positive duplex real-time RT-PCR result in 95% of samples). The coefficients of variation of the intra- and inter-assay reproducibility for CVA 6 and CVA 10 were both lower than 2%. Our results showed that this duplex real-time RT-PCR assay was a simple, rapid, and cost-effective method for simultaneous identification of CVA6 and CVA10.