Objective To establish and apply an effective PCR assay for detection of the Tupaia (tree shrew) adenovirus (TAV).Methods According to NCBI Genbank, TAV genome DNA from 19418 to 19917 were synthetized and inserted into a plasmid as positive standards. One pair of primers was designed based on this sequence. Sixty blood samples and fifty-six stool samples from tree shrew were detected with this PCR assay.Results A PCR method for detection of TAV was successfully established, with a high specificity and the sensitivity was 13.5 ×10^-7 μg/mL. The PCR results of testing sixty tree shrew blood DNA samples were negative. 24 positive cases were tested among 56 stool DNA samples. Sequencing of the samples confirmed a 42.9% infection rate of TAV in tree shrew stool samples, well consistent with the PCR results. Conclusions The PCR method for detecting TAV established in this study has good specificity and high sensitivity, therefore, can be used in conventional detection of tree shrew adenovirus.