To investigate the optimal doses of X-ray irradiation and plasmid injection in the anti-tumor effect of the pEgr-IFN γ gene–radiotherapy in vivo, we observed the anti-tumor effect of the pEgr-IFN γ gene–radiotherapy with different doses of X-ray irradiation and plasmid injection in mice with B16 melanoma and detected the concentration of IFN γ in peripheral blood by ELISA. The results showed that the tumor growth rate of the mice irradiated locally with 20Gy X-rays after the plasmid injection was significantly lower (,p,<,0.001) than that of the mice irradiated locally with 2Gy or 10Gy X-ray from the third day after irradiation and the mean survival period was longer (,p,<,0.001). The results also showed that the tumor growth rate of the mice injected locally with 20μg or 30μg plasmid was significantly lower (,p,<,0.05—0.001) than that of the mice injected locally with 10μg plasmid and the mean survival period was longer (,p,<,0.01). It was found that the concentration of IFN γ in peripheral blood of the mice treated with gene-radiotherapy was higher than that of the mice of plasmid-treated group (,p,<,0.05,p,<,0.05) or control group (,p,<,0.01,p,<,0.05) on the first day and the third day after irradiation. These results suggested that the optimal dose of X-ray irradiation of the pEgr-IFN γ gene–radiotherapy in vivo was 20Gy and the optimal dose of plasmid injected into tumors was 20μg. By inducing the higher expression of IFN γ gene in the tumor, the pEgr-IFN γ gene–radiotherapy could increase the concentration of IFN γ in peripheral blood, so as to enhance the body’s immunologic function and anti-tumor ability.