Abstract: Objective To establish and evaluate a single-tube multiplex real time RT-PCR assay for detecting rotavirus group A and astrovirus simultaneously. Methods The primers and probes were designed according to the conserved genome sequence of the 2 viruses mentioned above. A multiplex real-time RT-PCR assay was established. The stability, specificity and sensitivity of the assay were evaluated. The fecal samples from 128 patients with viral diarrhea were detected by the established assay and the results were compared through gene sequencing. Results The established multiplex real-time RT-PCR assay was specific for both rotavirus group A and astrovirus. The stability test showed the co-efficient variables was all less than 2.0% in 2 different samples. The detection limit of this assay was 101 copies/l for rotavirus group A and 102 copies/l for astrovirus in one reaction. Among the 128 clinical samples detected, 3.1% were astrovirus RNA positive and 18.0% were rotavirus group A RNA positive respectively. The positive samples were verified by sequencing. Conclusion The single-tube multiplex RT-real time PCR assay, established in this study for detecting and identifying rotavirus group A and astrovirus, is rapid, specific and sensitive and can be used in clinical etiological diagnosis and epidemiological investigation.