Objective To develop a rapid, sensitive and specific assay based on Taqman Probe real-time PCR to quantitate the proviral load of simian immunodeficiency virus (SIV). Method A 796bp specific fragment was amplified with RT-PCR, then cloned into pMD19T vector to construct a recombinant plasmid pMD SlVgag,which was used as SIV proviral DNA standards of this QPCR method. The QPCR system was optimized by using serial dilution of standards,and the sensitivity, specificity, repeatability and quantitation range of the method were also evaluated. Result The quantitative standard curve from 107 copies/well to 102 copies/well serial diluted plasmid DNAs showed that they had good linear correlation, the lowest limit of the standard curve reached 2x102 copies/well, the correlation (r>0.999). And coefficient of variability from 25 samples was below 1% (CV<1%). Conclusion The method showed high sensitivity, specificity and stability and will be used to quantify the SIV proviral DNA.