The active sites of a mixed Cu/Ce material and the doping effect of typical element (iron, Fe) on the active species and the catalytic behavior of Cu/Ce for CO preferential oxidation in rich H2 (CO-PROX) were investigated by in situ diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), in situ oxygen storage capacity measurement (OSC) combined with designed temperature-programmed reduction (TPR), along with Raman, X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and temperature-programmed desorption/reduction of CO (CO-TPD/TPR). These results showed that two kinds of surface active center were involved in the CuCe- and Fe-doped CuCe systems, that is, Cu+ as adsorption sites for the chemisorption and the activation of CO molecules, the surface reactive oxygen (the highly dispersed oxygen and surface lattice oxygen) that directly participated in the whole CO oxidation process. The addition of Fe into CuCe sample resulted in the incorporation of Fe into CeO2 lattice forming Fe–O–Ce structure and generated more oxygen vacancies, which not only enhanced the interaction between Cu and Ce to form more Cu+ absorption sites but also trapped the gas-phase oxygen and promoted the release of subsurface lattice oxygen to supply more reactive oxygen. Thus, the turnover frequency (TOF) value was increased from 3.62 × 10–2 s–1 for CuCe to 4.50 × 10–2 s–1 for Fe-doped CuCe. Moreover, with the enhancement of the lattice oxygen migration combined with the promotional role of Fe on the water gas shift (WGS), the capacity of the resistance to CO2 and H2O was enhanced for Fe-doping CuCe, and the corresponding stability time was largely prolonged from 170 to 400 h, in the coexistence of CO2 and H2O.