Abstract: Currently, low enzyme activity and high raw material cost seriously restricted the development of the industrial production of protease. In this study, transparent circle and shaking flask fermentation methods were used to screen out a neutral protease-producing strain, which was identified as Bacillus velezensis by morphological observation, physiological and biochemical results and 16S rDNA analysis, and named ppr3. The medium composition and fermentation conditions for this strain to produce neutral protease were optimized by one-at-a-time strategy and response surface methodology. The results showed that: The fermentation temperature was 33 ℃, inoculation amount was 8.5% (v/v), initial pH was 5.7, lactose 5.55 g/L, rapeseed 31.89 g/L, yeast extract 7.09 g/L, tween-80 0.03%, MgSO₄ 0.04%, K₂HPO₄ 0.04%, the protease activity was 500 U/mL. Compared with before optimization, it increased by 132%. The addition of rapeseed meal as nitrogen source to the medium significantly reduced the fermentation cost and provided a new way for the reuse of agricultural and sideline products, which had major implications in protease industrial fermentation.