Vinyl-functionalized silica spheres (VFSS) were synthesized through the one-step method. The effects of reactant ratio, temperature, reaction time and ammonium hydroxide dripping speed were investigated. The diameter of the spheres reached sub-2 μm magnitude by controlling the reaction conditions, which is suitable for high performance liquid chromatography (HPLC) packing material. The vinyl groups in the silica spheres were further successfully modified by octadecyl (C18) groups through a "thiol-ene" click reaction. Raw VFSS and C18modified VFSS (C-VFSS) were used as HPLC stationary phases to separate benzene homologs (benzene, toluene, and meta-xylene) and nonpolar compounds with different ring contents (benzene, phenantherene and fluoranthene). Both VFSS and C-VFSS exhibited hydrophobic retention behavior. Additionally, vinyl groups in the silica spheres may offer a π-π electron-donor-acceptor (EDA) retention mechanism for chromatographic separation.