Human enterovirus 71 (EV71) is the major etiological agent of hand, foot, and mouth disease (HFMD), which is a common infectious disease in young children and infants. EV71 can cause various clinical manifestations and has been associated with severe neurological complications; it has resulted in fatalities during recent outbreaks in Asian-Pacific regions since 1997. The early and rapid detection is critical for prevention and control of EV71 infection, since no vaccine or antiviral drugs are currently available. In this study, a simple and sensitive reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid detection of EV71. The detection limit of the RT-LAMP assay was approximately 0.01 PFU per reaction mixture, and no cross-reactive amplification with other enteroviruses was observed. The assay was evaluated further with 40 clinical specimens and exhibited 92.9% sensitivity and 100% specificity. This RT-LAMP assay may become a useful alternative in clinical diagnosis of EV71, especially in resource-limited hospitals or rural clinics of China and other countries in the Asian-Pacific region.