The major challenges in gene therapy for brain cancer are poor transgene expression due to the blood-brain barrier (BBB) and neurologic damage caused by conventional intracerebral injection. Non-viral gene delivery using ultrasound-targeted microbubbles (MBs) oscillation via the systematic transvascular route is attractive, but there is currently no high-yielding and targeted gene expression method. In this study, we developed a non-viral and angiogenesis-targeting gene delivery approach for efficient brain tumor gene therapy without brain damage. We developed a VEGFR2-targeted and cationic microbubbles (VCMBs) gene vector for use with transcranial focused ultrasound (FUS) exposure to allow transient gene delivery. The system was tested in a brain tumor model using the firefly luciferase gene and herpes simplex virus type 1 thymidine kinase/ganciclovir (pHSV-TK/GCV) with VCMBs under FUS exposure for transgene expression and anti-tumor effect. In vitro data showed that VCMBs have a high DNA-loading efficiency and high affinity for cancer cells. In vivo data confirmed that this technique enhanced gene delivery into tumor tissues without affecting normal brain tissues. The VCMBs group resulted in higher luciferase expression (3.8 fold) relative to the CMBs group (1.9 fold), and the direct injection group. The tumor volume on day 25 was significantly smaller in rats treated with the pHSV-TK/GCV system using VCMBs under FUS (9.7 ± 5.2 mm3) than in the direct injection group (40.1 ± 4.3 mm3). We demonstrated the successful use of DNA-loaded VCMBs and FUS for non-viral, non-invasive and targeted gene delivery to brain tumors.