The transcriptional factor Oct-4 and Survivin are the key regulatory factors in cancer cell proliferation and mitosis. A dual cancer-specific shRNA adenovirus vector, Ad5-Dual-shRNA, targeting Oct-4 and Survivin genes was constructed by molecular cloning and recombination. After cells were infected with virus, hepatocellular carcinoma cell line EHBH-H1 was used for detecting the expression of Oct-4 and Survivin proteins by Western blotting. The viral cytotoxic effect on cancer cells was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay in vitro, and the inhibition effect on tumor xenografts was observed in nude mice. The results showed that the expression of Oct-4 and Survivin in cancer cell line EHBH-H1 could be silenced markedly by Ad5-Dual-shRNA. In MTT and animal experiments, Ad5-Dual-shRNA also represented much stronger anti-tumor effect on tumor growth than Ad5-Surv-shRNA and Ad5-Oct4-shRNA. From this research we can draw a conclusion that the cancer-specific adenovirus vector expressing dual-shRNA targeting Oct-4 and Survivin genes may provide us a more effective, specific and convenient gene therapy method.