Objective To develop RT-PCR for detection of TMEV and apply the method. Methods To design specific primers on the basis of GD VII(GI:62039) genome sequences published in NCBI and establish RT-PCR. To verify the sensitivity and specificity of method after optimizing PCR. We infected 9 BALB/c mice intracerebrally and collected brain, heart, liver, spleen, lung, kidnet, cecal contents and serum samples the 6^th day postinfection. The samples were tested by the TMEV RT-PCR.100 mouse cecal contents samples were also detected to apply the established method. Results The 371bp single band was amplified using GDVII as template. Sensitivity test showed that the RT-PCR method can detect as low as 0.69 pg/μL GDVII cDNA. There were no objective band amplified when encephalomyocarditis virus, lymphocytic choriomeningitis virus, Japanese B encephalitis virus, murine norovirus and normal mouse brain tissue were used as case-control. All infected mice showed symptom of different degrees such as depression and hind limb paralysis the 3th day postinoculation and two of infected mice died the 5th day postinoculation. Tissues such as heart, liver, spleen, lung, kidney, brain, cecal contents and serum were collected and tested for TMEV. All the brain samples were detected positive for GDVII and other tissues were all negative; The 100 cecal contents samples were tested and all were negative. Conclusions RT-PCR for TMEV GDVII strain can detect virus infection in mouse tissues efficiently and can be used as a powerful supplement for the national standard of lab animal.