Recently, substantial mortality of farmed and wild tilapia caused by tilapia lake virus (TiLV) infection has been observed worldwide. However, sensitive and reliable diagnostic method is limited. A reverse transcription-loopmediated isothermal amplification (RT-LAMP) assay has been applied for the detection of TiLV nucleotide sequence. Six primers targeting two locations on the target gene based on a highly conserved sequence in the segment 1 (S1) region of the TiLV genome have been designed. The optimized RT-LAMP reaction was maintained at the isothermal condition of 63°C for 45 min. And the amplifications could be verified by turbidity or a colour change with the addition of SYBR Green I. Subsequently, RT-LAMP products could be observed by a ladder pattern following gel electrophoresis. The species-specific assay showed that the method was sensitive enough to detect as low as 1.6 copies of viral particle, and the assay was highly specific because no cross-reactivity was observed with other pathogens, and had a diagnostic sensitivity and specificity of 100% when TiLV-positive samples and non-target virus were tested. In summary, all the results demonstrate that this RT-LAMP is a rapid, effective and sensitive method for TiLV detection in tilapia aquaculture.