Abstract: According to conservative sequence of canine distemper virus nuclear proteins gene, a pair primers and TaqMan probe were designed, respectively. The standard recombinant plasmids for N gene were constructed as reference standard used in absolute quantification assay. The reaction conditions of FQ-PCR were optimized. The sensitivity of assay was tested with ten fold serial dilution of CDV and compared with ordinary RT-PCR. The quantitative curve was established using reference standard plasmids. The clinic samples were detected after specificity assay. The results demonstrated that the plasmid for FQ-PCR was favorable. The regression coefficient of the quantitative curve was 0.994. The detection sensitivity of FQ-PCR was 10 TCID50 and the specificity was determined by testing five other specimens, all of which yielded negative results. The detection system based on real-time RT-PCR was rapid, sensitive and steady, which could be used to detect the clinic samples.