Photocatalytic O2 activation to generate reactive oxygen species is crucially important for purifying organic pollutants, yet remains a challenge due to poor adsorption of O2 and low efficiency of electron transfer. Herein, we demonstrate that ultrafine MoOx clusters anchored on graphitic carbon nitride (g-C3N4) with dual nitrogen/oxygen defects promote the photocatalytic activation of O2 to generate ·O2− for the degradation of tetracycline hydrochloride (TCH). A range of characterization techniques and density functional theory (DFT) calculations reveal that the introduction of the nitrogen/oxygen dual defects and MoOx clusters enhances the O2 adsorption energy from −2.77 to −2.94 eV. We find that MoOx clusters with oxygen vacancies (Ov) and surface Ov-mediated Moδ+ (3 ≥ δ ≥ 2) possess unpaired localized electrons, which act as electron capture centers to transfer electrons to the MoOx clusters. These electrons can then transfer to the surface adsorbed O2, thus promoting the photocatalytic conversion of O2 to ·O2− and, simultaneously, realizing the efficient separation of photogenerated electron–hole pairs. Our fully-optimized MoOx/g-C3N4 catalyst with dual nitrogen/oxygen defects manifests outstanding photoactivities, achieving 79% degradation efficiency toward TCH within 120 min under visible light irradiation, representing nearly 7 times higher activity than pristine g-C3N4. Finally, based on the results of liquid chromatograph mass spectrometry and DFT calculations, the possible photocatalytic degradation pathways of TCH were proposed.