Interleukins as immunomodulators are promising therapeutic agents for cancer therapy. Previous studies showed that there was an improved antitumor immunity in tumor-bearing mice using recombinant Newcastle disease virus carrying for interleukin-2. Interleukin-12 is a promising antitumor cytokine too. So we investigated and compared the antitumor effect of genetically engineered Newcastle disease virus strains expressing both interleukin-12 and/or interleukin-2 (rClone30–interleukin-2, rClone30–interleukin-12, and rClone30–interleukin-12–interleukin-2). In vitro studies showed that rClone30s could efficiently infect tumor cells and express interleukin-12 and/or interleukin-2. 3-(4,5-Dimethylthiazol-2-y)-2,5-diphenyl-tetrazolium bromide results showed rClone30s possessed strong cytotoxic activities against multiple tumor cell lines (U251, HepG2, A549, and Hela). Animal studies showed that rClone30–interleukin-12–interleukin-2 was more effective in inhibition of murine hepatoma carcinoma tumors, with the mean tumor volume (day 14) of 141.70 mm3 comparing 165.67 mm3 of rClone30–interleukin-12 group, 210.47 mm3 of rClone30–interleukin-2 group, 574.70 mm3 of rClone30 group, and 1206.83 mm3 of phosphate-buffered saline group. Moreover, the rClone30–interleukin-12–interleukin-2 treated mice secreted more interferon γ (333.518 pg/mL) and its downstream cytokine interferon-γ induced protein 10 (16.006 pg/mL) in tumor than the rClone30–interleukin-12 group (interferon γ: 257.548 pg/mL; interferon-γ induced protein 10: 13.601 pg/mL), rClone30–interleukin2 group (interferon γ: 124.601 pg/mL; interferon-γ induced protein 10: 9.779 pg/mL), or rClone30 group (interferon γ: 48.630 pg/mL; interferon-γ induced protein 10:1.650 pg/mL). For the survival study, rClone30–interleukin12–interleukin2 increased the survival rate (12 of 16) of the tumor-bearing mice versus 11 of 16 in rClone30–interleukin–12 group, 10 of 16 in rClone30–interleukin-2 group, 7 of 16 in Clone30 group, and 0/16 in phosphate-buffered saline group, respectively. To determine whether the mice treated with recombinant virus developed protective immune response, the mice were rechallenged with the same tumor cells. The results showed that viral-treated mice were significantly protected from rechallenge. These results suggest that expressing both interleukin-2 and/or interleukin-12 could be ideal approaches to enhance the antitumor ability of Newcastle disease virus, and rClone30–interleukin-12–interleukin-2 is slightly superior over rClone30–interleukin-12 and rClone30–interleukin-2 alone.