Abstract: The fermentation medium composition of L.Lactis KLDS4.0325 isolated from self- made koumiss Xinjiang were optimized for bacteriocin production by single factor experiment( carbon source,nitrogen source,different mixed nitrogen sources),Plackett- Burman,The steepest grade test and Box- Behnken design double- plate method.The inhibitory zone diameter( mm) was used as evaluation index and quantified against Escherichia coli ATCC 25922.PB Experiment results showed that sucrose,peptone and sodium ascorbate had significant influence on bacteriocin production.And finally the optimal combination of the medium constituents for bacteriocin production was determined as: 128.99 g / L sucrose,17.59 g / L peptone,10 g / L casein peptone,10 g / L K₂HPO₄,60 g /L Ca CO₃,1.5 g /L Mg SO₄·7H₂O,1.49 g / L sodium ascorbate,1.5 g / L β- glycerophosphate,CH₃ COONa·3H₂O,Na Cl,Mn SO4 respectively 1.0 g / L.Under the optimal culture condition,the inhibitory zone diameter was 24.76 mm and the bacteriocin inhibitory activity was increased by 3.31 times and reached up to 5000 U / m L.Meanwhile,the consistent results between the prediction and experiments indicated the established model in this study is feasible.