Abstract: Xanthohumol was separated and purified by macroporous adsorption resin from waste hops (Humulus lupulus L.) , and its inhibition activity against DNA oxidative damage was investigated. The resulted showed that HPD100 A was appropriated to purify xanthohumol from waste hops, with adsorption rate was 100% and desorption rate was 77.69%. The optimal conditions for purification were as follows:the loading concentration of xanthohumol was 0.25mg/m L, the loading volume was 15 BV, the p H of loading solution was 5.5, the loading rate was 2BV/h, the elution was 95% ethanol, the elution rate was 2BV/h and the elution volume was 3BV. Under the optimized conditions, the purity of xanthohumol in extract was increased from 4.95% to 51.50%, with the recover rate was 72.56%. The purified xanthohumol was evaluated its inhibition activity against DNA oxidative damage. The result showed that the purified xanthohumol had dramatically inhibition activity against DNA oxidative damage induced by Phen-Cu SO4-VC, and its inhibition activity (EC50=0.22mg/m L) significantly better than that of ascorbic acid (EC50=0.51mg/m L) and rutin (EC50=0.93mg/m L) .