Abstract: The chiral membrane of amylose-tris-(3, 5-dimethylphenylcarbamate)-polyethersulfone was prepared by phase conversion method using the amylose-tris-(3, 5-dimethylphenylcarbamate) as the material. The chiral membrane chromatographic separation of propranolol hydrochloride and metoprolol were studied by using the self-made chiral membrane chromatographic device combined with a high performance liquid chromatograph. The effects of injection volume, flow rate and membrane size on the separation of membrane were studied. Under the optimal conditions, the separation factors (α) and resolution (Rs) with water as mobile phase were 3.00 and 0.95 for propranolol hydrochloride, 1.65 and 0.46 for metoprolol, respectively. The study opens up a new way for the isolation and analysis of chiral compounds.