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The TiO2-H2O2 system possesses excellent oxidation activity even under dark conditions. However, the mechanism of this process is unclear and inconsistent. In this work, the binary component system containing TiO2 nanoparticles (NPs) with single electron-trapped oxygen vacancy (SETOV, VO·) and H2O2 exhibit excellent oxidative performance for tetracycline, RhB, and MO even without light irradiation. We systematically investigated the mechanism for the high activity of the TiO2-H2O2 under dark condition. Reactive oxygen species (ROS) induced from H2O2 play a significant role in improving the catalytic degradation activities. X-ray photoelectron spectroscopy (XPS) and electron paramagnetic resonance (EPR) results firstly confirm that H2O2 is primarily activated by SETOVs derived from the TiO2 NPs through direct contribution of electrons, producing both ·O2-/·OOH and ·OH, which are responsible for the excellent reactivity of TiO2-H2O2 system. This work not only provides a new perspective on the role of SETOVs playing in the H2O2 activation process, but also expands the application of TiO2 in environmental conservation.