Background: TheWest Nile Virus (WNV), discovered in New York, USA in 1999 after it was first isolated in Uganda in 1937, has since spread not only in the United States but also around the world. Africa, Eurasia, Australia, and the Middle East have sporadic cases of the disease. Objectives: We aimed to find real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to be more sensitive than conventional RT-PCR, and more rapid and efficient than conventional RT-PCR and real-time RT-PCR forWNVdetection. Methods: A total of 32 genomic sequences from different strains of WNV were analyzed to identify conserved nucleotide sequence regions. Six WNV specific RT-LAMP primers targeting the E gene were designed. Results: The novel primer for the real-time RT-LAMP assay can detect WNV with high specificity. The efficiency of the real-time RTLAMP assay is higher than the conventional RT-PCR and real-time RT-PCR. Real-time RT-PCR and conventional PCR require at least 30 – 40 min and 2 h, respectively, to yield results, whereas real-time RT-LAMP provides positive results in only 10 – 20 min. Conclusions: The novel primers were developed by analyzing of 32 genomic sequences of WNV strains. The primers were designed from the most conserved region of the E gene for real-time RT-LAMP. The LAMP assay is a rapid, efficient, highly sensitive, and specific tool for the identification of WNV.