In this work, monodisperse organosilane hybrid polymer microspheres with a particle size of about 5µm were synthesized using seed swelling polymerization. The organosilicon reagent methacryloxypropyltrimethoxysilane was introduced into the polymer framework as a copolymerization monomer, and the crosslinking degree of the microspheres was improved by the hydrolysis-condensation reaction of siloxanes. The synthesized hybrid microspheres have good mechanical strength as well as low swelling, with swelling propensity values of 0.167 and 0.348 in methanol and acetonitrile, respectively. Hybrid microspheres modified with cysteine have a hydrophilic interaction chromatography/reversed-phase liquid chromatography mixed-mode retention mechanism. Compared to the commercial cysteine-modified silica column, the synthesized stationary phase has higher separation selectivity for partially acidic or basic samples and better basic resistance for use under high pH mobile phase conditions (at least 10).