Oncolytic virotherapy is being developed as a promising platform for cancer therapy due to its ability to lyse cancer cells in a tumor-specific manner. Vaccinia virus has been used as a live vaccine in the smallpox eradication program and now is being potential in cancer therapy with a great safety profile. Vaccinia strain Guang9 (VG9) is an attenuated Chinese vaccinia virus and its oncolytic efficacy has been evaluated in our previous study. To improve the tumor selectivity and oncolytic efficacy, we here developed a thymidine kinase (TK)-deleted vaccinia virus based on Guang9 strain. The viral replication, marker gene expression and cytotoxicity in various cell lines were evaluated; antitumor effects in vivo were assessed in multiple tumor models. In vitro, the TK-deleted vaccinia virus replicated rapidly, but the cytotoxicity varied in different cell lines. It was notably attenuated in normal cells and resting cells in vitro, while tumor-selectively replicated in vivo. Significant antitumor effects were observed both in murine melanoma tumor model and human hepatoma tumor model. It significantly inhibited the growth of subcutaneously implanted tumors and prolonged the survival of tumor-bearing mice. Collectively, TK-deleted vaccinia strain Guang9 is a promising constructive virus vector for tumor-directed gene therapy and will be a potential therapeutic strategy in cancer treatment.