Abstract: Extraction of flavonoids from Opuntia milpa alta peel can make full use of its flavonoids resource and exploit activity component. Effects of ethanol concentration, extraction temperature, extraction time and ratio of solvent to material on flavonoids yield were analyzed with response surface methodology. The results show that regression equation fit well with experimental data, the optimum procedure of flavonoids extraction are: ethanol concentration of 67%, extraction temperature of 76℃, extraction time of 5.7 h, ratio of solvent to material of 21.4 mL/g and that the flavonoids yield is up to 6.45% with one-time extraction. Primary flavonoids glycosides in Opuntia milpa alta peel are mainly isorhamnetin 3-O-(2′,6′-dirhamnosyl) glucoside and isorhamnetin 3-O-(6′-rhamnosyl) glucoside.