CO oxidation, one of the most important chemical reactions, has been commonly studied in both academia and the industry. It is one good probe reaction in the fields of surface science and heterogeneous catalysis, by which we can gain a better understanding and knowledge of the reaction mechanism. Herein, we studied the oxidation state of the Cu species to seek insight into the role of the copper species in the reaction activity. The catalysts were characterized by XRD, N2 adsorption-desorption, X-ray absorption spectroscopy, and temperature-programmed reduction. The obtained results suggested that adding of Fe into the Cu/Al2O3 catalyst can greatly shift the light-off curve of the CO conversion to a much lower temperature, which means the activity was significantly improved by the Fe promoter. From the transient and temperature-programmed reduction experiments, we conclude that oxygen vacancy plays an important role in influencing CO oxidation activity. Adding Fe into the Cu/Al2O3 catalyst can remove part of the oxygen from the Cu species and form more oxygen vacancy. These oxygen vacancy sites are the main active sites for CO oxidation reaction and follow a Mars-van Krevelen-type reaction mechanism.