This data article contains data related to the research article entitled "Rapid detection of enterovirus and Coxsackievirus A10 by a TaqMan based duplex one-step real time RT-PCR assay" (Chen at al., 2017) [1]. Primers and probe sequence design are among the most critical factors in real-time polymerase chain reaction (PCR) assay optimization. Linearity, sensitivity, specificity and precision are the crucial criteria which are used to evaluate the performance of a new method. This data article report the primers and probe design and precision assessment of the new assay. VP1 gene of Coxsackievirus A10 (CV-A10) and 5'-NCR of different enterovirus (EV) serotypes were retrieved from GenBank database and aligned. The intra- and inter-assay variation were assessed using high, medium and low concentration of control plasmid DNA and viral RNA samples.