Glycyrrhizic acid (GL) and its derivants, glycyrrhetinic acid 3-O-mono-β-d-glucuronide (GAMG) and glycyrrhetinic acid (GA) hydrolyzed in subcritical water, are bioactive substances and edulcorators. In this work, a separation strategy for these three substances was established. The effects of adsorbent and eluent were investigated by static/dynamic adsorption and multi-stage desorption with the mechanism analysis. The adsorption of them onto EXA50 resin was well fitted by the pseudo second-order kinetic model. The optimal dynamic adsorption flow rate was 6 bed volume (BV)/h, and water of pH = 12 was used to elute GL at 4 BV/h, then n-buthanol was used subsequently to elute GA at 1 BV/h, and finally 90% ethanol was applied to elute GAMG at 2 BV/h. As a result, purities of these compounds increased, which demonstrated that this adsorption-desorption technology was simple and efficient, and indicated the potential for large-scale purification and preparation of GL and its derivants in the future.