Nattokinase was purified from unclarified Bacillus subtilis culture filtrate using an expanded bed with a purification factor of 8.2 and at a yield of 95%. The optimal pHs for adsorption and elution were 6.0 and 7.0, respectively. The expanded bed route shortened the process time by 8-10 h and increased the yield by 50% when compared with the conventional method.