Supported copper on silica catalysts prepared by sol-gel and impregnation methods were studied in this paper. The surface structures of these catalysts were characterized by various techniques, including BET, XRD, FTIR, XPS, TPR, and ESR. The results showed that the distribution of copper species was in different ways in the catalysts prepared by the two methods. Cu(II) species highly dispersed in the silica matrix for the sol-gel catalyst, while copper oxide clusters are dominant in the CUO-SiO2 sample prepared by the impregnation method. The catalysts were then used for dehydrogenation of 2-butanol. Obvious differences of catalytic behavior were observed for the catalysts prepared by the two methods. High selectivity (>90%) toward dehydrogenation and high 2-butanol conversion was observed for the impregnated catalyst; however, for the CUO-SiO2 sol-gel catalyst, very low dehydrogenation selectivity and 2-butanol conversion were obtained. The surface structures of catalysts were closely related to the preparation methods, and the catalytic behaviors were affected subsequently. The copper oxide clusters, which may be reduced to Cu-0, are responsible for the dehydrogenation reaction. The highly dispersed Cu(II) ions were inactive for catalyze 2-butanol dehydrogenation.