Abstract: Objective To construct the bicistronic eucaryotic expression vector of human interleukin 12（hIL12）pVAX1-IRES-hIL12 co-expressing the hIL12 double subunits. Methods The fusion gene P35-F2A-P40,amplified by overlap extension PCR,was inserted into the downstream of the eukaryotic expression vector pVAX1-IRES.Then the recombinant plasmid pVAX1-IRES-hIL12 was transfected to 293T cells,and its expression was detected by ELISA. Results Enzyme digestion and sequence analysis showed that the bicistronic eucaryotic expression vector pVAX1-IRES-hIL12 was successfully constructed.The expression of recombinant plasmid was detected by ELISA,suggesting that the fusion genes can express in 293T cells. Conclusion Successful construction of the vector can provide an effective immune tool for the development of anti-tumor vaccine.