Objective To develop a RT-PCR method for determination of Mammalian Orthoreovirus 3 (Reo3) in laboratory animal and the biological materials or the biological products for human use from the animal. Methods Two pair of primers specific to the sequences of M1 gene were designed according to the published Mammalian Orthoreovirus 3.With which the RNA of Reo3 was extracted and reversely transcribed to cDNA as a template for PCR amplification.The developed RT-PCR method was optimized, verified for specificity and sensitivity. Results The developed RT-PCR method is good in ensitivity, specificity and stability, and its minimum detection limit using the recombinant plasmid containing Reo3 gene as atemplate was 0.42pg/μL,and the lowest detection virus titer is 10-9/mL. Conclusion The developed RT-PCR method can be used in detecting the Mammalian Orthoreovirus 3 (Reo3) in laboratory animaland the biological materials or the biological products for human use from the animal.