Objective To establish an accurate TaqMan probe real-time quantitative PCR (qPCR)method for detection of Theiler's-like virus of rats (TLV). Methods Primers and TaqMan probes specific to 3622~3729 nt region were designed according to the whole genomic sequence of TLV representative strain. Using a synthesized plasmid as DNA standard template, the stability, specificity, and sensitivity of the qPCR method were determined. Results In the standard curve, R² value was 0.99 with a high specificity. The sensitivity of the real-time PCR was less than 10 copies/μL, which was 100 times higher than the ordinary PCR method. No cross reactions appeared to the other rat viruses. Conclusions The TaqMan probe qPCR method established in this study has advantages such as simple to use, high sensitivity and specificity.