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Enterovirus 71 (EV71) is a major pathogen of hand, foot, and mouth disease (HFMD). To date, no antiviral drug has been approved to treat EV71 infection. Due to the essential role that EV71 3 C protease (3C^pro) plays in the viral life cycle, it is generally considered as a highly appealing target for antiviral drug development. In this study, we present a transgene-encoded biosensor that can accurately, sensitively and quantitatively report the proteolytic activity of EV71 3C^pro. This biosensor is based on the catalyzed activity of a pro–interleukin (IL)-1β-enterovirus 3C^pro cleavage site-Gaussia Luciferase (GLuc) fusion protein that we named i-3CS-GLuc. GLuc enzyme is inactive in the fusion protein because of aggregation caused by pro–IL-1β. However, the 3C^pro of EV71 and other enteroviruses, such as coxsackievirus A9 (CVA9), coxsackievirus B3 (CVB3), and poliovirus can recognize and process the canonical …