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CO catalytic oxidation over Pd–Cu/Al2O3 catalysts calcined at different temperatures was studied. It was found that Pd–Cu/Al2O3-573 catalyst showed the best performance and stability than those of Pd–Cu/Al2O3-473 and Pd–Cu/Al2O3-673 catalysts. The presence of Cu2Cl(OH)3 played vital roles in achieving the highest performance over the Pd–Cu/Al2O3-573 catalyst. The preserved surface carbonate species over the Pd–Cu/Al2O3-473 catalyst and the absence of the Cu2Cl(OH)3 species over the Pd–Cu/Al2O3-673 catalyst accounted for their slight lower performances. High moisture and CO2 played negative effects on CO conversion. The former was coming from the competitive adsorption of gaseous water that hindered CO activation and benefited to the formation of carbonates under the saturated moisture conditions, and the latter might be originated from the competitive adsorption and the covering of carbonates on the catalyst surface.