Abstract: In order to improve the yield of nattokinase and promote its industrial production and application, Bacillus subtilis was isolated from fresh natto from different producing areas, and the strain with larger ratio of hydrolysis circle to colony was selected through preliminary screening of colony morphology and re-screening of casein plate, and the nattokinase activity in its fermentation broth was measured, and a strain X₃ with relatively high enzyme activity was selected. X₃ strain was identified as Bacillus subtilis by systematic 16S rDNA sequencing and phylogenetic tree comparison. On the basis of single factor experiment, response surface methodology was designed to optimize the liquid fermentation conditions of nattokinase in shake flask, and the optimum conditions for liquid fermentation of X₃ strain were determined as follows: temperature 34 ℃, initial pH6.5, inoculation amount 2%, bottling amount 20%, fermentation time 30 h. Under these conditions, the enzyme activity of nattokinase produced by fermentation was 393.095 U/mL, which was higher than that of nattokinase purchased. This experiment laid a certain foundation for the subsequent industrial production.