In the present study, the performance and adsorption characteristics of five macroporous resins for the separation of glabridin from Glycyrrhiza glabra L. have been evaluated. The adsorption and desorption properties of glabridin on macroporous resins including HPD100, HPD300, HPD800, NKA and H103 were compared. HPD100 resin offered the best adsorption and desorption capacities based on the research results. Both Langmuir and Freundlich isotherms were used to describe the interactions between the solutes and resins at different initial concentrations. Dynamic adsorption and desorption experiments on HPD100 resin packed column were conducted to optimize the separation process of glabridin from licorice extracts. After the treatment with stepwise elution on HPD100 resin, the content of glabridin in the product increased from 0.21% to 32.2% which is 153-fold higher than it in G. glabra L. roots and the recovery yield was 79.7%. The results indicated the good ability of HPD100 resin for separation glabridin and the study may provide scientific references for the large-scale glabridin production from G. glabra L. or other plants extracts.