Objective To establish a quick and accurate method for detection of tree shrew adenovirus (TAV) using TaqMan real-time fluorescence quantitative PCR. Methods Based on the published TAV genome sequence, a 3^, conserved sequence was used to design specific probe primers. A standard curve was prepared using a recombinant plasmid containing the target gene fragment. A real-time fluorescence quantitative PCR method was established for detecting TAV based on TaqMan probe. Results The detection method was specific and was not cross-reactive with other common pathogens. The detection limit of the method was 3.7 copies/ μL, showing a high sensitivity. The correlation coefficient was 0.998, and the efficiency was 95.7%. The amplification result showed a fine linear relationship, and the repeatability test effect was good. Conclusions The TAV real-time quantitative PCR detection method based on TaqMan probe has been successfully established. It has high sensitivity and reproducibility and can be applied to early detection of TAV infection.