Several hundred U mL-1 of Nattokinase (NK), a fibrinolytic enzyme, can be produced by culturing recombinant Bacillus subtilis in Luria-Bertani broth in a shaking flask. For use as a nutraceutical, large-scale preparation and a simple purification process is required. The present study utilized a fed-batch process with a pH-stat and low-glycerol-level-maintain feeding strategy to cultivate a B. subtilis strain carrying a pHT01 plasmid with an NK-encoding gene (B. subtilis/pHT01-aprN1). Finally, a NK activity of 7778 ±17.28 U mL-1 was obtained, which represented a 26-fold increase of NK activity by high cell density cultivation compared to the flask culture. Furthermore, fermentation supernatant was successively purified by ammonium sulfate precipitation and nickel column affinity chromatography with a total NK recovery rate of 65.2%.