Objective： To investigate the optimization of fermentation conditions of a novel neutral protease produced by Bacillus cereus. Methods： The Plackett- Burman design was firstly used to screen the key factors influencing the activity of the novel neutral protease produced by Bacillus cereus， and then their optimal levels were further determined through the Box-Behnken response surface experimental design. Results： The main factors affecting the activity of the novel neutral protease of Bacillus cereus were glucose， peptone and pH. The compositions of the optimized fermentation medium and culture conditions were： glucose 35 g/L， peptone 40.38 g/L， pH 7.15， sodium chloride 15 g/L， magnesium sulfate 1.5 g/L， Tween-80 10 g/L， loaded volume 50 mL/250 mL， cell age 12 h， and fermentation time 36 h. Under the above conditions， the activity of the novel neutral protease produced by Bacillus cereus was up to 696.51 U/mL， a 1.93-fold increase than that in the original conditions. Conclusion： The results provide a good basis for industrial production and application of the novel neutral protease.