Abstract: In order to establish a differential diagnosis Real-time RT-PCR with highly sensitivity, specificity and repeatability, a pair of primers and TaqMan probe were designed according to characteristics of cleavage site sequences of F gene of genotype ⅤⅡ Newcastle disease virus strains in this study. The standard curve was established by using cRNA standard prepared in vitro transcription as positive template, the related indexes were tested. The results showed that this assay has a good effect of differential detection of genotype ⅤⅡ NDV; 102 copies·μL^-1 of template RNA could be detected at least. The sensitive of this assay was 100 times higher than conventional RT-PCR. The real-time fluorescent quantitative RT-PCR for detection of genotype ⅤⅡ Newcastle disease virus was established. This assay could be used as differential diagnosis method of genotype ⅤⅡ NDV strains, which couldn’t be identified by conventional methods. Our results indicated that the method established in this study can be used as rapid identification assay for the detection of genotype ⅤⅡ NDVs.