Micrometer-sized mesoporous SBA-15 silica spheres have been synthesized using triblock copolymer poly(ethyleneoxide)-block-poly(propyleneoxide)-block-poly(ethyleneoxide) (EO20PO70EO20) as structure-directing agent with the assistance of 1,3,5-trimethylbenzene and KCl. By controlling the composition of synthesis gel, SBA-15 silica spheres with particle size in the range of 2-4.5 mu m could be facilely obtained. The obtained SBA-15 silica spheres were characterized by X-ray diffraction, scanning electron microscopy, and N-2 physisorption technique. Dimethyloctadecylchlorosilane modified mesoporous SBA-15 silica with diameter of 4.5 mu m was explored as stationary phase in high-performance liquid chromatography, and its chromatographic characteristics for separation of some small molecules and relatively large bio-molecules have been studied. (C) 2009 Elsevier Inc. All rights reserved.