Cutinase is a kind of multifunctional hydrolase, which has important application value in the food processing and textile refining industry. The recombinant expression system of Escherichia coli (E.coli) BL21 (DE3) was used to achieve the recombinant expression of Humicola insolens cutinase in E.coli, and the activity of extracellular supernatant in shaking flask level reached 170 U/mL. The recombinant cutinase was characterized. The optimum pH was 8.5, the optimum temperature was 80 ℃, with good pH stability and temperature stability. In order to further improve the possibility of the industrial application of H.insolens cutinase, the recombinant E.coli was cultured in 3 L fermentor and the cultivation condition is optimized. The optimum fermentation conditions: maintain pH 7.0 and 37 ℃ in early culture phases about 12～14 h; when the cell concentration OD₆₀₀ up to 50, fermentation entered induced phases，adjust the temperature to 30 ℃, 0.2 g/(L·h) lactose solution were added with constant flow rate. The total fermentation period was about 36 h. The highest activity of enzyme reached to 2 233 U/mL, which is 13 times about the enzyme activity in shake flask level.