To evaluate the effect of PCR enhancer on molecular identification of Chinese herbal medicine, and select the optimal enhancers suitable for traditional Chinese medicine (TCM), genomic DNA from 180 kinds of Chinese herbal medicine was extract by CTAB method and alkaline lysis method, respectively. PCR success rate of five universal fragments (ITS2, psbA-trnH, rbcL, matK, trnL-trnF) was compared in a PCR system with and without enhancer. PCR efficiency of Real-time PCR was also compared in a PCR system with and without enhancer. Results showed that PCR success rate of ITS2,psbA-trnH, rbcL fragment was increased by using PVP and BSA. The PCR efficiency was decreased by PCR enhancer in Real-time PCR system. The results indicate that BSA and PVP as PCR enhancer can dramatically increase PCR success rate and genotyping accuracy in TCM molecular authentication.