Objective The anti-tumor effect by sequential treatment with Newcastle disease virus (NDV) strain 7793 and 5-FU in liver-metastases mice model was evaluated and immune-active response stimulated by sequential therapy was investigated. Methods Liver metastasis mice model was established by intra-peritoneal injection. The model mice were randomly divided into 3 groups, being given PBS (0.1 mL/d,10 d), NDV7793 [512 HU/(kg·d),5 d] and NDV7793[512 HU/(kg·d),5 d] + fluorouracil [5-FU,10 mg/(kg·d),5 d]. The effect on survival time,body weight,liver weight change and the formation of liver metastasis in tumor-bearing mice model were detected after different treatments in evaluating the regression of mice liver metastasis by sequential therapy. The detection of thymus index and IFN-γ concentrations in liver tissue of tumor-bearing mice model may indicate the stimulation of immune-active response by sequential therapy. Results The mean survival time of tumor-bearing mice treated with NDV7793 and 5-Fu sequentially was 32 d , which was significantly higher than those of tumor-bearing mice treated with NDV7793(30 d) or PBS injections (17 d), respectively (P< 0.05); The metastatic foci of tumor-bearing mice treated with NDV and 5-FU sequentially (30.60 ± 9.32) which was significantly less than those of tumor-bearing mice treated with PBS injection (273.30 ± 30.73), (P <0.05), seem quite similar to those treated with NDV (24.83 ± 6.90),(P > 0.05), and the liver weight was lighter than PBS (P < 0.05); Compared with NDV treatment, the decreased thymus index and increased amount of the effector IFN γ were observed in tumor-bearing mice treated with NDV 7793 and 5-FU sequentially (P <0.05). Conclusions The sequential therapy with Newcastle disease virus 7793 strain and 5-FU was observed to co-exert a significant suppressive effect in liver metastases of colon cancer cells in tumor-bearing mice model. Compared with NDV treatment , the survival time of mice model and the induction of antitumor effector molecules were significantly improved after sequential therapy.