OBJECTIVE In order to enhance vaccine response, we constructed a dicistronic expression plasmid containing double HBsAg immunogenes. METHODS At first, pcDNA3.1 plasmid vector was digested with NheI and EcoRI to get the coding sequence of the small (S) surface protein of HBV, then cloned into pCI-neo vector and named it pCI-S. By PCR amplification, the product of IRES-S was digested with SalI & BamHI, and cloned into pBluescript IIK+S to generate pBKS-IRES-S vector, then subcloned to the pCI-S plasmid to generate pCI-S-IRES-S, which is a dicistronic plasmid of double value HBsAg genes. RESULTS Two plasmids we constructed were digested with related restriction nucleic enzymes. Sequence analysis of HBsAg and IRES-S gene did not reveal any mutation. CONCLUSIONS The construction of dicistronic plasmid of divalue HBsAg immunogenes has been well cloned, which is convenient for further research on cell expression and gene immunization in animals.