Abstract: A leucine aminopeptidase producing strain Bacillus subtilis ZH-Zj016 was obtained by protoplast transformation technology. Fermentation conditions of the strain were firstly optimized through single factor experiments. Then a series of fermentation control strategies, such as the minimum dissolved oxygen control, temperature-shift culture and cell permeability regulation, were performed and the optimal fermentation conditions for 15L fermenter was confirmed as follows:medium volume of 8L, initial pH8.0, 7‰ of inoculum volume, agitation rate of 250⁵00r/min pressure at 0.06⁰.08MPa, 40% dissolved oxygen (DO) upholding (The DO and agitation rate were coupled) , ventilation volume of 1.2vvm, culture temperature at 39℃ within 15h and to 37℃ thereafter. After fermentation for 28h, leucine aminopeptidase activity produced by the strain reached 138U/mL, which was 21 times of the original strain.