Tumor hypoxia is a significant factor leading to the resistance of tumors to treatment, especially for photodynamic therapy and radiotherapy where oxygen is needed to kill cancer cells. Oxygen delivery agents such as oxygen-saturated perfluorocarbon nano-emulsions and lipid oxygen microbubbles have been employed to supply oxygen to hypoxic tumors with ultrasound activation. Such oxygen delivery systems are still associated with several drawbacks, including premature oxygen release and the dependence of external stimuli. To address these limitations, we developed oxygen nanobubbles that were enclosed by the acetalated dextran polymer shells for spontaneous oxygeneration in response to a minor pH drop in the tumor microenvironment. The acetalated dextran polymer shell serves as a robust barrier against gas dissolution in the circulating blood to retain the majority of the oxygen payload, and its pH-responsive property enables an abrupt burst release of oxygen in the mild acidic tumor microenvironment. The acetalated dextran oxygen nanobubbles exhibited excellent stability and biocompatibility. In vitro and in vivo experiments were conducted to investigate the pH-responsive oxygen release. The external stimuli-free supply of oxygen by the acetalated dextran oxygen nanobubbles was evaluated on CNE2 tumor-bearing mice, and the intratumoral oxygen level increased by 6-fold after the administration of the oxygen nanobubbles, manifesting that our pH-responsive oxygen nanobubbles hold great potential as a potent oxygen delivery agent to overcome the hypoxia-induced resistance.