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Natto has attracted worldwide attention because of its health benefits and long history in Japanese food. It has been found that a potent fibrinolytic enzyme named nattokinase, which is extracted from natto, is able to prevent atherosclerosis. The production of nattokinase may be influenced by various factors such as temperature, shaking speed, volume of medium, fermentation time and so forth. Three‐step response surface methodology was applied to obtain the optimal operation conditions of the fermentation process in order to maximize the nattokinase yield. The three major steps are described as follows. First, the important factors for fermentation were identified by L8 orthogonal array experiment. The chosen factors were temperature (37 or 45C), shaking speed (110 or 150 rpm), volume of medium (80 or 120 mL), Brix of wheat bran extract (1.5 or 3°), Brix of soy meal extract (1 or 2°), glucose concentration (0.6 or 1.2%) and fermentation time (24 or 36 h). Second, a regression equation was established between the response (i.e., the enzyme activity) and the two statistically significant factors (i.e., the volume of medium and fermentation time). Third, the optimal solutions for the volume of medium and fermentation time were obtained based on the response surface of the regression equation. According to the response surface analysis, the optimal operation conditions for the fermentation process should be 80 mL and 37.0817 h for the volume of medium and the fermentation time, respectively, which resulted in 459.11 FU/mL as the predicted enzyme activity.