Immunotherapy is an important cancer treatment strategy; nevertheless, the lack of robust immune cell infiltration in the tumor microenvironment remains a factor in limiting patient response rates. In vivo gene delivery protocols can amplify immune responses and sensitize tumors to immunotherapies, yet non-viral transfection methods often sacrifice transduction efficiency for improved safety tolerance. To improve transduction efficiency, a strategy employing low ultrasound transmission frequency-induced bubble oscillation is optimized to introduce plasmids into tumor cells. Differential centrifugation isolated size-specific microbubbles (MBs). The diameter of the small MB population is 1.27 ± 0.89 µm and that of the larger population is 4.23 ± 2.27 µm. Upon in vitro insonation with the larger MB population, 29.7% of cancer cells are transfected with DNA plasmids, higher than that with smaller MBs (18.9%, P <0.05) or positive control treatments with a commercial transfection reagent (12%, P < 0.01). After 48 h, gene expression increases more than twofold in tumors treated with large, as compared with small, MBs. Furthermore, the immune response, including tumor infiltration of CD8+ T cells and F4/80+ macrophages, is enhanced. It is believed that this safe and efficacious method can improve preclinical procedures and outcomes for DNA vaccines in cancer immunotherapy in the future.