Plackett-Burman design, steepest ascent design and response surface methodology were used to optimize fermentation mediumof L-lysine. Plackett-Burman designmethod was applied to evaluate the main significant factors, and the result had shown that sodium chloride、magnesium sulfate and corn steep liquor were the major factors. Then, the Box-Behnken design and response surface methodology we used to confirm the optimum parameters with NaCl 2.72%, MgSO₄·7H₂O 0.05%, and corn steep liquor (CSL) 21.13 g/L, which were proceeded based on thegreatest response area of NaCl, MgSO₄·7H₂O and corn steep liquor. Under the optimal conditions, the theoretical yield was gotten of L-lysine of 104.75 g/L, but the experimental yield was 104.60 g/L that was increased 18.96% level than before optimization.