Abstract: The extraction of total flvonoids from mulberry leaves with Tween-80 synergistic method was researched in this paper.Using the single factor experiment and orthogonal test to decide the method that in the Tween- 80synergistic method extract total flvonoids from mulberry leaves, and also compare it with the extraction without the surfactant.The results showed that in the extraction process about Tween- 80 synergistic method Tween- 80 's mass concentration was 1%, volume of solvent was 125mL, temperature was 50℃, extraction time was 1.5h. And with this method, the extraction yield of toal flavonoids from mulberry leaves was 2.467%, and in the same condition, without the Tween-80, the yield of it was 1.718%.Thus Tween-80 increase the yield of total flavonoids from mulberry leaves significantly.