Catalytic oxidation of toluene over noble metal catalysts is a representative reaction for elimination of volatile organic compounds (VOCs). However, to fully understand the activation of molecular oxygen and the role of active oxygen species generated in this reaction is still a challenging target. Herein, MgO nanosheets and single-atom Pt loaded MgO (Pt SA/MgO) nanosheets were synthesized and used as catalysts in toluene oxidation. The activation process of molecular oxygen and oxidation performance on the two catalysts were contrastively investigated. The Pt SA/MgO exhibited significantly enhanced catalytic activity compared to MgO. The oxygen vacancies can be easily generated on the Pt SA/MgO surface, which facilitate the activation of molecular oxygen and the formation of active oxygen species. Based on the experimental data and theoretical calculations, an active oxygen species promoted oxidation mechanism for toluene was proposed. In the presence of H₂O, the molecular oxygen is more favorable to be dissociated to generate •OH on the oxygen vacancies of the Pt SA/MgO surface, which is the dominant active oxygen species. We anticipate that this work may shed light on further investigation of the oxidation mechanism of toluene and other VOCs over noble metal catalysts.