To investigate the process of separating and purifying total flavonoids from Smilax glabra.With the contents of total flavonoids, astilbin and engelitin as indices, the optimum process of separating and purifying flavonoids from Smilax glabra was screened by static and dynamic adsorption tests.The static saturated adsorption capacity of polyamide resin to flavonoids of Smilax glabra was 144 mg/g (saturated in 2 h). The dynamic adsorption capacity was 128.125 mg/g, the optimum conditions of elution were as follows: the adsorbed resin was washed by 20BV water firstly, then by 20BV 95% ethanol. The content of obtained total flavonoids reached 71.84%, as the content of astilbin was 14.32%, and the content of engelitin was 1. 08%.The total flavonoids of Smilax glabra is able to be easily separated and purified by polyamide resin under the optimum conditions above.