Osteosarcoma has been known as a type of aggressive bone cancer that is characterized by a high inclination for pulmonary metastasis. Despite the current therapeutic regimes including surgical excision of primary tumor, chemotherapy and advanced radiotherapy, patients often suffer a high incidents of the neoplasm recurrence[1]. Therefore, understanding the molecular regulation mechanisms for this cancer and exploration of novel treatment strategies such as immunotherapy have been carrying out. The purposes of this research were to determine the relationship of microRNA-21 and development propensity of VEGF and CD133 positive osteosarcoma cells. Besides, the study also focused on immunotherapy feasibility of interleukin-21 plasmid vector transfected into NIH3T3 fibroblast cells with high-performance reagents in a mouse model. Experimental molecular biology methods were performed to confirm the blockage of miR-21 has more effect on low-VEGF expression tumor cells, whereas inhibition of VEGF receptor has more effect on high-VEGF expression tumor cells. Besides, IL-21 incorporated plasmid vector can be successfully transfected into mouse fibroblast with Lipofectamine 2000 and 3000. Moreover, an OS immunocompetent animal model was established and ready to be used for IL-21 immunotherapy in vivo study.