The composition of hesperidin hydrolysate is complicated, including saccharides, hesperetin and hesperetin-7-O-glucoside（HMG）. HMG from hesperidin hydrolysate was separated and purified by six macroporous adsorption resins and high performance semi-preparative liquid chromatography. The structure of the product was characterized by IR, UV-Vis and XRD analysis. And the antioxidant activities of HMG, hesperidin and hesperetin were compared by hydroxyl radical scavenging test. The results showed that HPD300 macroporous resin was the most suitable resin for the purification of HMG from enzymatic hydrolysis solution. The static and dynamic total adsorption amounts of HPD300 resin to the main components of enzymatic hydrolysis solution were 48.35 mg/g and 35.82 mg/g, respectively，which were mainly used for separation of sugar impurities. The purity of HMG in the eluent was increased from 80.37% to 97.83% by using 10BV, 45% ethanol as eluent with the flow rate of 1.2 mL/min. The final purity of monomer HMG, was 98.21% after further separation and purification by semi-prepared liquid phase. The product was verified as HMG by UV-Vis, IR and XRD spectra. And the hydroxyl radical scavenging capacity of HMG was slightly higher than those of hesperetin, much higher than that of hesperidin.