Abstract: The extraction process of total flavonoids from mulberry leaves was optimized based on antibacterial activity. The curde extract obtained at the optimal condition was fractionated by different solvents and then qualitatively analyzed. The contents of total flavonoids of fractionated extracts were determined. The diameter of antibacterial circle and minimum inhibitory concentration of extracts on Escherichia coli (Ec.) and Staphyloccocus aureus Rosenbach (Sa.) were investigated. The stability of the better fractionated extract was tested. The results showed that the optimal extraction condition for curde extract is 70 ℃, 70 % ethanol concentration, 4 h and solid-liquid 1:30(g:mL). The fractionated extract being better antibacterial effect was ethyl acetate extract. Furthermore, ethyl acetate extract had better stabilities on temperature and UV irradiation.