[Abstract] Objective To establish a real-time RT-PCR method for detection of simian parainfluenza Virus 5 type (SV5) in biological material from monkeys and explore its application．Methods According SV5 virus NP gene fragments, a pair of specific primers and TaqMan probe were designed and synthesized; SV5 RNA standards were prepared using some molecular biological methods. Then the linearity, sensitivity, specificity and stability of the method were measured. Last, the method was used for testing lung samples of 24 monkeys. Results we established successfully SV5 real-time PCR method. The linear range of 6.8 ?109 copies/μL ~ 6.8 ?105 copies/μL, a sensitivity of 6.8 copies/μL, coefficient of variation (CV) was 0.63% ~ 8.71%, inter-assay CV 1.52% - 12.38%, and the method is high specific. There wasn’t detected positive in lung samples of 24 monkeys. Conclusions Establishment of the method provides the SV5 quantitative detection assay for the experimental animals and animal-derived biological products.