A high performance liquid chromatographic (HPLC) method using amylase chiral stationary phase for the separation of bisoprolol enantiomers has been developed. Tris (3,5-dimethylphenyl carbamate) amylase chiral stationary phase was used in the normal-phase mode. The effects of the concentration of organic solvents (iso-propanol and ethanol), flow rate and column temperature on the enantiomer separation were studied systematically. The best separation was obtained with hexane-ethanol-diethyl amine (88 : 12 : 0.1, v/v/v) as mobile phase at 20 degrees C. The optimal flow rate was 0.6 mL/min. The detection wavelength was set at 270 nm. By using this method, the enantiomers of bisoprolol can be separated quickly and easily.