The influence of the order of component introduction and Mo/Fe ratio in the silica-supported Mo-Fe-O catalysts on chemical and phase composition, redox and acid properties of the active catalyst surface, and catalytic activity in the selective oxidation of propylene glycol is considered. The catalysts are synthesized by wetness impregnation with a co-presence of the Fe and Mo components in the solution and sequential impregnation with varying the order of component introduction. The samples are characterized by low-temperature adsorption of N-2, UV-vis, Raman spectroscopy, TPR-H-2, and pulse NH3 adsorption methods. The effect of the preparation method on the phase composition and catalytic properties in selective oxidation of propylene glycol is shown. The catalyst prepared by sequential impregnation technique is more active and selective as compared to the one prepared by co-impregnation technique. The maximal selectivity towards methylglyoxal is achieved over the catalyst 3MoO(3)/Fe2O3/SiO2 prepared by introduction of the Mo-containing component onto the surface of the FeOx-precovered support. The role of the FeOx species adsorbed on the support surface with the participation of the SiOH groups is connected with the ability to facilitate the formation of highly dispersed Fe-2(MoO4)(3) and MoOx nanoparticles that are strongly bound with the SiO2 surface.