Regulating the binding energy between the catalysts and reaction intermediate species is crucial for electrocatalytic processes. As according to the Sabatier principle, excessively strong binding energies can be detrimental. The presence of oxygen vacancy defects is beneficial, leading to a relatively lower binding energy. In this study, ZnO catalysts with varying oxygen vacancy contents were successfully synthesized with different solvents and hydrothermal treatment temperatures. The oxygen vacancy content of prepared samples was characterized using X-ray photoelectron spectroscopy and electron paramagnetic resonance, while the electrocatalytic CO 2 reduction reaction (CO 2 RR) experiments were performed in a flow cell. A proportional relationship was observed between the CO production rate and oxygen vacancy content. As a result, the catalyst with a hydrothermal temperature of 180 °C for 3 h (ZnO-EG-180-3) exhibited the highest oxygen vacancy content, accompanied by a high CO yield. The sample was stable in CO 2 RR for over 50 h at a constant current of 150 mA, maintaining a stable CO faradaic efficiency (FE) exceeding 90%, and the CO yield was more than 2625 μmol• h −1 •cm −2 , while lower than 10% for H 2 . Density functional theory calculations confirmed that the presence of oxygen vacancies in ZnO catalysts reduces the binding energy of the *CO intermediate, facilitating *CO desorption, mitigating the poisoning effect of *CO on active sites, and promoting the generation of CO generation.