Abstract: The expression conditions of lipase from genetically engineering Pichia pastoris were optimized and its high-density fermentation in a 10 L fermenter was investigated. The results showed that the optimal pH was in range of 5.5-6.0, suitable methanol dosage was 1%, the optimal fermentation time was 96 h after induction with methanol and temperature was 28-30 ℃, and it was also indicated that 2% of corn steep powder could substitute yeast extract and peptone as nitrogen source. By using glycerol feeding strategy with DO control in high-density fermentation, the dry cell weight was up to 120.1 g/L, and the total protein concentration reached at 2.5 g/L in the fed batch fermentation phase. 54 hours after the induction with methanol, the lipase activity got to be 564 μmol/(min·mL), which is as much as two times of the lipase activity in the shake flask fermentation.