To investigate the preparation method and application of immobilized betulinic acid (BA) derivatives in monolithic chromatography columns.The monolithic column poly (GMA-EDA-BA-co-EDMA) was prepared with a one-pot in situ copolymerizations of the ligand ethylenediamine-betulinic acid (EDA-BA),the monomer glycidyl methacrylate (GMA),the cross-linker ethylene glycol dimethacrylate (EDMA),the porogen (n-propanol/1,4-butanediol),and initiator 2,2′-azobisisobutyronitrile (AIBN),under optimized polymerization conditions.The columns were characterized by elemental analysis,scanning electron microscopy,and micro-HPLC.The result showed that most of the tested proteins and enzymes,including trypsin,bovine albumin,human serum albumin,and cytochrome C,have no retention in this column apart from α-glucosidase,which the resulted monolithic column had a selective affinity to α-glucosidase.It was shown that the "one-pot" method can be well applied to the preparation of monolithic columns for the immobilization of natural molecules.