The separation of amino acid (AA) enantiomers shows significancefor chemistry, food, and biology, but remains challenging due to their similar properties. A promising nanoporous chiral covalent organicframework (COF) as a stationary phase for high-performance liquid chromatography (HPLC) suffers from the irregularity and widely distributedparticle size of the chiral COF. Herein, we show the facile preparation of a chiral COF-monolith as a stationary phase for HPLC enantiomericseparation of AAs via orthogonal experiments. The CTzDa-monolith is prepared by the incorporation of the model chiral COF named CTzDa intothe porous poly(ethylene dimethacrylate-co-methacrylate) monolith and reveals great permeability and mechanical stability. The correspondingCTzDa-monolithic column gives better chiral HPLC separation of AAs than the commercial Poroshell 120 chiral-T column. Thermal dynamicanalysis and molecular docking calculations imply the involvement of stereoscopic hydrogen, π-π, and van der Waals interactions between theCTzDa and AAs during HPLC enantioseparation. The facile incorporation of the chiral COF into the porous monolith will promote the potential ofa chiral COF as a stationary phase for HPLC.