Abstract: Used the adsorption rate,desorption rate and recovery rate as indexes,on the basis of single factor experiment,orthogonal design was used to optimize the technological conditions of purifying total flavonoids from leaves of Ilex centrochinensis S. Y. Hu by D101 macroporous resin. The antibacterial activity in vitro of total crude and purified flavonoids was studied by oxford cup test. The optimum technological conditions for purifying total flavonoids from leaves of Ilex centrochinensis S. Y. Hu by D101 macroporous resin are as follows:Total flavonoids concentration was 0.735 mg/mL,pH was 1.93,sample volume was 3 BV,diameter-height ratio was 1:8.5,adsorption velocity was 6 BV/h. It was stand still for 30 minutes before desorption,then 3 BV deionized water was used to remove impurities. Desorption ethanol(8 BV)concentration was 70%,desorption velocity was 6 BV/h. The recovery rate of total flavonoids was 91.06%,the mass fraction of total flavonoids was increased from 48.52% to 78.26%. The results of bacteriostasis in vitro showed that:The antimicrobial circles of total flavonoids from leaves of Ilex centrochinensis S. Y. Hu against Staphylococcus aureus,Escherichia coli,Pseudomonas aeruginosa and Shigella sonnei were 14.07,17.15,7.12 and 7.57 mm,respectively. Except Escherichia coli,the antibacterial effect against the other 3 kind of bacteria of crude extract was better than the purified producton.