In order to develop a real-time fluorescent reverse transcription loop-mediated isothermal amplification（RT-LAMP）assay for detection of duck Tembusu virus（DTMUV），one set of special primers were designed based on the highly conservative sequence of DTMUV NS5 gene，the reaction time and temperature were optimized，followed by evaluation on specificity，sensitivity，repeatability and clinical applicability. The results showed that the optimal conditions for the established method were 65 ℃ for 25 minutes，only DTMUV could be specifically amplified，and no reaction with other avian viruses was observed；its minimum detection limit was 4×102 copies/μL，which was consistent with qRT-PCR and 100 times higher than that of conventional RT-PCR；with high intra and inter-assay repeatability，the method was applied to detect the liver samples from artificially infected ducks at different doses，the positive detection rates of the samples vaccinated with the challenge doses of 103.7 TCID50 and 102.7 TCID50 were 100%，that of those with 101.7 TCID50 was 90%（9/10）. In conclusion，the established method was with high specificity，repeatability and sensitivity，and could be used for rapid diagnosis of DTMUV.