Metal-organic frameworks (MOFs), defined as a class of microporous hybrid materials, are established by coordination of metal cations with functional organic ligands. In addition to outstanding porosity and large surface area, the structures and functions of MOFs can be designed and adjusted based on different destinations, which would be employed as stationary phases in various chromatographic modes. Pepsin, a class of protein, has been investigated as chiral selector owing to their unique interactions with analytes based on chiral or affinity selectivity. In this work, MOF-5 was exploited to grow on the support of poly (glycidyl methacrylate)-co-(ethylene dimethacrylate) [poly(GMA-co-EDMA)] monoliths by layer-by-layer self-assembly method. Pepsin was subsequently bonded with the carboxyl group of MOF-5 through amidation reaction. The ultimate pepsin@MOF-5@poly(GMA-co-EDMA) column was applied for enantioseparation of six basic chiral drugs by capillary electrochromatography with good resolution and repeatability. Compared with the pepsin modified monolithic column, the newly prepared column with MOF-5 shows significantly enhanced enantiomeric resolution, which reveals that MOFs-modified capillary monolithic columns lead a promising road to separation of racemates by chromatography.