The encoding gene lks2 of lumbrokinase was cloned from Eisenia foetida and successfully expressed in Pichia pastoris GS115 with an activity of 254 U/mL. Optimization of culture conditions in shake flask showed that the best induction conditions were as follows: initial OD600=1.5, pH 5.5, temperature 28 ℃. Under these conditions, the activity of lumbrokinase peaked 397.6 U/mL at 84 h. Moreover, the amplification culture of recombinant P. pastoris GS115-pPIC9K-lks2 was occurred in a 10 L fermenter, of which the optimized inoculation concentration was 9±0.5 g/L. Under this high-density fermentation condition, the pre-culture time was shortened by 11.5 h and the activity of lumbrokinase was raised to 1 098.2 U/mL, which has a considerable potential for industrial application. In this study, the yield of lumbrokinase from P. pastoris was effectively increased by the optimization of induction conditions and the high-density fermentation, laying a foundation for the industrial production and application of lumbrokinase.