AIM: To design and establish a method of multiplex PCR normalization for simultaneously detecting HBV and HCV.METHODS: Two pairs of primers with a 20 bp joint sequence were used to amplify the target genes of HBV and HCV by two rounds of amplification. After the two rounds of amplification all the products had the joint sequence. Then the joint sequence was used as primers to finish the last amplification. Finally multiplex PCR was normalized to a single PCR system to eliminate multiplex factor interference. Four kinds of nucleic acid extraction methods were compared and screened. A multiplex PCR normalization method was established and optimized by orthogonal design of 6 key factors. Then twenty serum samples were detected to evaluate the validity and authenticity of this method.RESULTS: The sensitivity, specificity, diagnostic index and efficiency were 83.3%, 70%, 153.3% and 72.2%,respectively for both HBsAg and anti-HCV positive patients,and were 78.6%, 80%, 258.6% and 79.2%, respectively for HBsAg positive patients, and were 75%, 90%, 165%and 83.3%, respectively for anti-HCV positive patients.CONCLUSION: The multiplex PCR normalization method shows a broad prospect in simultaneous amplification of multiple genes of different sources. It is practical, correct and authentic, and can be used to prevent and control HBV and HCV.