We designed two pairs of primers and their corresponding TaqMan probes according to gH, gE gene of PRV. By optimizing the probe’s concentration, Mg2+ concentration, primers concentration and sample DNA extraction, rea1-time fluorescent quantitative PCR (FQ-PCR) which can quickly identity field virus and vaccine virus of PRV was established. According to our results, the dynamic range of the FQ-PCR assay is between 10×101 copies/mL and 10×108 copies/mL, and the detection limit of FQ-PCR is 1.0×101 copies/mL, which is 100 fold higher than that of conventional PCR. We detected 60 doubtful tissue samples using the FQ-PCR assay, serum neutralization and conventional PCR. In conclusion, the FQ-PCR method is rapid, sensitive, specific and accurate, and can be used to detect field strains of PRV rapidly. The closed-tube format of the assay minimized the risk of contamination of subsequent reaction and the assay can be performed in 2 h or less. Development of real-time quantitative PCR provides the basis for the early and rapid detection and analyzing quantitatively the infectious degree of PRV.