Summary A sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid detection of Tembusu virus (TMUV) infection. The reaction was performed in one step in a single tube at 64°C for 45 min, with SYBR Green I dye added prior to amplification. The detection limit of the RT-LAMP assay was approximately 10 copies/μl, and no cross-reaction with other avian viruses was observed. The assay was evaluated further for the diagnosis of TMUV in field samples and compared with conventional RT-PCR, demonstrating that results of the RT-LAMP assay corresponded to those of conventional RT-PCR. In conclusion, RT-LAMP with SYBR Green I dye was shown to be a sensitive, simple assay for the rapid diagnosis of TMUV infection in ducks.