Through introducing octakis (3-mercaptopropyl) octasilsesquioxane (POSS-SH) synthesized in our lab to the prepolymerization solution containing stearyl acrylate (SA), 1,6-hexanediol ethoxylate diacrylate (HEDA) in the existence of porogenic solvents (tetrahydrofuran, 1,4-butanediol and 1-propanol), a POSS-containing hybrid monolithic column was fabricated via photo-initiated thiol-acrylate polymerization within 7 min. The resulting poly(SA-co-HEDA-co-POSS) monoliths were investigated by physical characterization and chromatographic evaluation. It was found that both the additive amount of thiol group and the proportion of porogenic solvents played vital effect on column efficiency, pore morphology and hydrophobicity of monolithic columns. Consequently, the poly(SA-co-HEDA-co-POSS) monolith possessed superior thermal stability, suitable permeability and homogeneous microstructure. The highest column efficiency was ∼111,000 N m−1 for butylbenzene at the linear velocity of 0.71 mm s−1 in reversed-phase liquid chromatography. Subsequently, baseline separations of 9 phenolic compounds, 5 anilines and 5 antibiotics were achieved, indicating the monolithic poly(SA-co-HEDA-co-POSS) column had great ability for separation of small molecules. The analytic results of the tryptic digest of BSA and HeLa were also proved that the hybrid monolith had potential for the analysis of complicated biological samples. Display omitted •POSS-SH was firstly employed to prepare hybrid monoliths.•A monolithic poly(SA-co-HEDA-co-POSS) column was fabricated via photo-initiated thiol-acrylate polymerization.•The poly(SA-co-HEDA-co-POSS) monolithic column exhibited high efficiencies in cLC.•The monolith was applied for separation of complicated biological samples in cLC-MS.