The effect of fed-batch fermentation on the production of nattokinase byBacillus subtilisLSSE-22 was investigated in shake flask and bioreactor. Glucose and soy peptone were identified as the optimal carbon source and nitrogen source through batch fermentation in shake flask, respectively. Furthermore, the effect of feeding substrate, feeding manner and feeding time on the production of nattokinase was studied on the basis of optimization of initial concentration of glucose and soy peptone. The results showed that the production of nattokinase was 1437.34 IU/mL, which was 21.38% higher than that by batch culture. In the 7.5 L bioreactor, the maximal production of nattokinase reached 2046.47 IU/mL, which was significantly higher than that by batch culture.