Molecularly imprinted polymer (MIP)-based chromatographic separation materials, owing to their advantages of unique selectivity, low cost, suitable reproducibility, and acceptable stability, have attracted a great deal of research in different fields. In this investigation, a new type of MIP-coated silica (MIP/SiO2) separation material was developed using sulfamethoxazole as a template; the specific recognition ability of MIP and appropriate physicochemical properties (abundant Si-OH, suitable pore structure, good stability, etc.) of SiO2 microbeads were combined. The MIP/SiO2 separation materials were characterized carefully. Then, various compounds (such as sulfonamides, ginsenosides, nucleosides, and several pesticides) were used to comprehensively evaluate the chromatographic performances of the MIP/SiO2 column. Furthermore, the chromatographic performances of the MIP/SiO2 column were compared with those of other separation materials (such as non-imprinted polymer-coated silica, C18/SiO2, and bare silica) packed columns. The resolution value of all measured compounds was more than 1.51. The column efficiencies of 13 510 plates per meter (N m-1) for sulfamethoxazole, 11 600 N m-1 for ginsenoside Rd, and 10 510 N m-1 for 2'-deoxyadenosine were obtained. The acceptable results verified that the MIP/SiO2 column can be applied to separate highly polar drugs such as sulfonamides, ginsenosides, nucleosides, and pesticides.