The production of lipase by recombinant Pichia pastoris was studied in 15 L fermenter. The single factor test was firstly carried out to optimize pH, aeration, inoculum, stirring speed and inducer concentration. The most important factors reflected from the single factor test were then selected for three levels orthogonal test in 700 L fermenter. The optimum fermentation conditions were pH 5.0, fermentation temperature 30 °C, stirring speed 250 r/min, inoculum volume 5%, dissolved oxygen 20%-30%, methanol 129 L and fermentation time 160-170 h. The results showed that the addition of 0.5% yeast extract (w/v) and 1% peptone (w/v) in 3 portions (once every 48 hours) after induction could significantly improve the lipase activity and the maximum enzyme activity reached 18 U/mL.