Enterovirus consists of a variety of viruses that could cause a wide range of illness in human. The pathogenesis of these viruses remains incompletely understood and no specific treatment is available. Better methods to study enterovirus infection in live cells will help us better understand the pathogenesis of these viruses and might contribute to antiviral development. Here in this study, we developed fluorescent cell-based reporter systems that allow sensitive distinction of individual cells infected with enterovirus 71 (EV71). More importantly, these systems could be easily used for live-cell imaging by monitoring viral-induced fluorescence translocation after EV71 infection. We further demonstrated that these reporter systems could be used to study other enterovirus-mediated MAVS cleavage and they are sensitive for antiviral activity testing. Therefore, integration of these reporters with modern image-based analysis has the potential to generate new insights into enterovirus infection and facilitate antiviral development.