The first human cases of infection with avian influenza A(H7N9) virus were reported in March 2013 in China. The number of confirmed cases continues to increase, although almost all the cases are limited to China. In this study, a one-step real-time RT-PCR assay was developed for detecting the novel A(H7N9) virus. This assay was shown to have high specificity, good linearity, and high sensitivity to a broad range of Eurasian H7 viruses. The assay is useful both for diagnostic purposes in cases of suspected human infection with the influenza A(H7N9) virus and in the surveillance of both avian and human influenza viruses. A diagnostic system using this assay was prepared at 74 prefectural and municipal public health institutes and 16 quarantine stations in Japan early into the human H7N9 infection outbreaks, enabling potential diagnoses of H7N9 infection across Japan.