Abstract: A high performance liquid chromatographic(HPLC)separation of tryptophan(Trp) enantiomers was successfully realized by using chiral crown etherCrownpak CR(+)as the immobile phase and aqueous perchloric acid solution mixed with methanol as the mobile phase. The influences of methanol content in the mobile phase，the flow flux of the mobile phase and the column temperature on the separation performance were investigated. The resolution ability can be improved respectively by increasing methanol content in the mobile phase in a certain extent，by decreasing the flux of the mobile phase or the column temperature. The retention time of samples can be significantly shorten by increasing methanol content in the mobile phase，by increasing the flux of the mobile phase or the column temperature. The optimal conditions for analysis and separation were established with methanol: perchloric acid=15:85 (V/V) as mobile phase at the flow flux of 0.8 mL/min，the U V wavelength of 220nm and the column temperature of 20℃. Under the above conditions，the retention time of samples was less than 12 min and the separation factor can reach 2.06.The proposed method had advantages of short analysis time，high resolution，high accuracy as well as high precision.