The key factors on high-level polygalacturonate lyase (PGL) production in recombinant Pichia pastoris were investigated. In 250 mL shake flask, the optimal glycerol concentration, initial methanol concentration, methanol supplementation quantity, duration of induction, initial pH, medium volume were 40 g/L, 3.1 g Methanol/g DCW, 0.51 g Methanol/g DCW (in every 24 h), 72 h, pH6.0 and 30 mL, respectively. In 7 L fermentor, constant glycerol feeding, constant and DO-stat methanol feeding strategies were applied to enhance cell density and PGL production. Finally, the dry cell weight was 80 g/L, and the maximum yield of PGL was achieved to 217 U/mL, increased by 66.2% than that of flask level.