Background: Oncolytic virus can specifically replicate in and then lyse tumor cells, but seldom in normal cells. Further studies have shown the significant therapeutic effect of oncolytic virotherapy combining with other strategies, such as chemo-, radio-, and immunotherapy et al. In this study, we investigated the combinational effect of oncolytic virus ZD55-TRAIL and chemotherapy drug doxorubicin (DOX) on human laryngeal squamous cell carcinoma (LSCC). Methods: The effect of ZD55-TRAIL combined with DOX on cell growth was assessed in LSCC Hep2 cells and normal cells by MTT assay. Hochest 33342 staining was performed to observe cell morphological changes. Western blot was used to detect the expression of apoptotic activation proteins. The in vivo antitumor efficacy of combination treatment was estimated in laryngeal cancer xenograft models. Results: The combination of ZD55-TRAIL and DOX exhibited enhanced inhibitory effects on laryngocarcinoma cell growth, and had few side effects to normal cells in vitro. Chemotherapy drug increased the inducement of tumor cell apoptosis mediated by oncolytic virus. In vivo experiment confirmed that the combination treatment significantly inhibited Hep2 laryngocarcinoma xenografts growth in mice. Conclusion: The oncolytic viro-chemotherapy is a potent therapeutic approach for in vitro cytotoxicity evaluation of Hep2 cells and xenograft growth in vivo.