Abstract: Objective To construct pTRKH2-PsT/CD and pTRKH2-PsT/UPRT dual suicide gene therapy system into tumor-hypoxia-targeting carrier of Infant Bifidobacterium. Methods CD gene and UPRT gene were amplified from pGEX/CD and pGEX/UPRT by PCR. The CD gene, UPRT gene were inserted into lactic acid expressing plasmid pTRKH2-PsT, then constructed two recombinant plasmids of pTRKH2-PsT/CD and pTRKH2-PsT/UPRT were introduced into Bifidobacterium using electroporation. The expression of CD and UPRT in Bifidobacterium were detected by RT-PCR or through SDS-PAGE and Silver staining. The killing effects of pTRKH2-PsT/CD and pTRKH2-PsT/UPRT suicide gene therapy system on Melanoma B16-F10 cells were measured using methyl thiazolyl tetrazolium (MTT) assay. Results The CD gene and UPRT gene was successfully recombined into plasmid pTRKH2--PsT. The sequencing results of CD gene and UPRT gene in dual suicide system are consistent with data in Genebank. Both mRNA and protein level of CD and UPRT were effectively expressed in gene recombinant Infant Bifidobacterium. The survival rate of Melanoma B16-F10 cells treated with CD/UPRT and 5-FC ranged from 1.7%~13.3%, while the rates in control, CD and UPRT groups ranged from 81.3%~91.7%,26.7%~58.3% and 78.3%~86.7%, respectively. Conclusion CD gene and UPRT gene were successfully inserted into pTRKH2-PsT vector and effectively recombined into Infant Bifidobacterium. This dual suicide gene therapy system showed a powerful killing efficiency on tumor cells.