By reversed-phase high-performance liquid chromatography, the extraction and separation of glabridin by from licoricce root was performed in this work. The column efficiencies and resolutions of glabridin were investigated with mobile phase composition on the reversed-phase chromatographic system. The glabridin collected from licorice root was identified by LC/MS. The mobile phase used to extract glabridin were composed of ethanol, methanol, acetone, and ethyl acetate. For one-hour ultrasonic extraction with solvent of ethyl acetate, the favorable content of glabridin was obtained as 1.26g/kg. The glabridin was well separated in the mobile phase composition of 50/50 vol. % (acetonitrile/water).