A novel hydroxyl functionalized organic–inorganic hybrid monolithic column for high performance liquid chromatography (HPLC) was synthesized viafree radical copolymerization with sonocation-assist, which could shorten the time of the sol–gel process. Vinyltrimethoxysilane (VTMS) was used as the monomer and vinyl ester resin was used as both the monomer and crosslinker. The conditions of preparation were investigated and the characteristics of the hybrid column were studied by SEM and Fourier transform infrared spectroscopy. The obtained column showed high permeability and low backpressure. The column was used to separate lysozyme from egg white in a short time (6 min) by HPLC, and benzene and its homologs were separated by the hybrid column. In addition, influences on the elution of lysozyme, such as the pH value and the buffer concentration, were studied. Additionally, the lysozyme separated by the hybrid column showed high biological activity, which was assayed by the method of turbidimetry.