MnOx and FeOx-based catalysts supported on γ-Al2O3 (0.1–20 wt %) were prepared by using two methods: incipient wetness impregnation and single-step flame spray pyrolysis. The effect of the structural properties and composition on the CO oxidation activity was systematically evaluated and correlated with the preparation methods. The characterization of the samples by XRD, X-ray absorption spectroscopy, TEM, and temperature-programmed reduction by hydrogen revealed that, in contrast to the use of incipient wetness impregnation, flame spray pyrolysis leads to the formation of highly dispersed homogeneously distributed FeOx and MnOx species. A partial incorporation of Fe and Mn ions into the γ-Al2O3 lattice for low metal oxide loadings and for samples prepared by flame spray pyrolysis was observed. In general, the CO oxidation activity increased with the transition metal oxide loading. Below 200 °C, Mn-based catalysts demonstrated the highest catalytic performance. However, the addition of water decreased the performance, especially at lower temperatures, which demonstrates the competitive adsorption on the active sites. The presence of NO had no effect on the CO conversion. A significant effect of the preparation method on the catalytic performance was observed during hydrothermal aging. The superior distribution of the active species obtained by flame spray pyrolysis leads to thermally more stable catalysts.