Objective To establish a reverse transcription nested polymerase chain reaction (RT-nPCR ) assay for detection of tree shrews orthoreovirus (TRV). Methods Three strains of TRV were respectively isolated from fresh feces of three tree shrews that came from the same field at different times. We designed and synthesized two pairs of MRV L1 gene nested primers and established the system of RT-nPCR. The TRV RNA was extracted and reversely transcribed to cDNA as a template for nested-PCR amplification. The developed RT-nPCR was optimized. The specificity and sensitivity were tested. Finally, the RT-nPCR was used to detect TRV in 25 tree shrew samples. Results Taking the genomic RNA of TRV as template, the RT-nPCR was able to amplify a specific fragment band targeting the L1 gene, while there were no target bands in the normal cell control, (Wa strain rotavirus, hepatitis A virus, and herpes simplex virus). The RNA of TRV was diluted by 1:10 to 1:10⁹. Each dilution sample was analyzed by the RT-nPCR. The minimum detectable concentration of RNA was 0.01 pg/μL. The results of RT-nPCR detection showed that 4 of the 15 tree shrews were TRV-positive in the survival group, and 10 of 10 tree shrews were TRV-positive in the death group. Conclusions The RT-nRCR assay established in this study is accurate, specific and sensitive. Therefore, it can be used for routine detection of TRV in quality assurance testing.