Oxygen vacancy acts an important role in adjusting the chemical properties of MnO. In this paper, two-dimensional MnO catalysts with different oxygen vacancy concentration are obtained by doping Cu. It is researched that the K species in the interlayer of birnessite-type MnO can be substituted during the Cu doping process. Meanwhile, this process will generate the oxygen vacancy. Interestingly, the formation of an appropriate numbers of oxygen vacancy in MnO distinctly enhances the low-temperature reducibility and oxygen species activity, which improves the catalytic activity for the toluene oxidation (T = 220 °C, E=43.6 kJ/mol). However, an excessive concentration of oxygen vacancy in MnO sample performs against the activity improvement for toluene oxidation. In situ DRIFTS are applied to elucidate the main intermediates and conversion pathway on MnO-OV3 with moderate concentration of oxygen vacancy. The results demonstrate that the adsorbed toluene can interact with oxygen species of catalyst to form physisorbed benzaldehyde, aldehydic adsorbate and benzoate species. In addition, it is found that the oxygen vacancy concentration plays an important effect on the oxidation of benzoate species owing to the acceleration effect of oxygen vacancy in the activation of gaseous oxygen.