Nattokinase is a potent fibrinolytic enzyme. The composition of the medium for nattokinase production was optimized respectively. Effect of glucose concentration on nattokinase activity and optimal glucose concentration in fermentation process were investigated. L₉ (3³) orthogonal array design was applied to investigate the effects of Na₂HPO₄ to NaH₂PO₄ ratio, CaCl₂, MgSO₄ on nattokinase activity and search for the optimal concentrations of the three inorganic salts. The results showed that Na₂HPO₄/NaH₂PO₄ was the main factor to affect nattokinase activity, then was CaCl₂ and MgSO₄ in turn. The optimum concentrations of liquid fermentation medium components were as followed: glucose 1% , Na₂HPO₄ to NaH₂PO₄ ratio 0.4% : 0.1% , CaCl₂ 0.01% and MgSO₄ 0.1%. Under the optimal conditions, the nattokinase activity was equivalent to 871.56 urokinase IU/mL. To mask the smelly flavor, the nattokinase powders were encapsulated. The research will be helpful to the development of nattokinase products.