As a non-invasive therapeutic, radiotherapy (RT) has been extensively used for solid tumor treatment. However, intratumoral hypoxia leads to severe RT resistance or failure. Moreover, damage from RT to normal tissues limits the application of high doses of radiation to eliminate cancer cells. Therefore, simultaneously improving the curative efficacy while minimizing the side effects of RT is in pressing need. Hence, the purpose of this study is to use oxygen-based microbubbles (O₂ @MBs) combined with ultrasound (US) targeting microbubble destruction (UTMD) technology to overcome hypoxia prior to RT, evaluate the effects of O₂ @MBs on contrast-enhanced ultrasound (CEUS) imaging enhancement, investigate the optimum delivery route of O₂ @MBs, and evaluate the therapeutic efficacy. In this study, O₂ @MBs were injected intravenously or locally and the distribution of O₂ @MBs in tumors or regions surrounding the tumors are compared by US imaging. The hypoxic status of tumors and their sensitivity to RT were investigated. Our findings suggest that O₂ @MBs combined with UTMD can significantly enhance the effects of RT. In addition, the in vivo biosafety assay demonstrates good biocompatibility, indicating great potential for clinical translation.