In order to enhance the concentration of ε-polylysine (ε-PL) with Streptomyces albulus ZC7, response surface methodology were used to optimize the fermentation medium. A Plackett-Burman design was used to evaluate the influence of eight factors firstly. Results showed that the concentration of glucose, yeast extract and (NH4)2SO4 played important roles in influencing the content of ε-PL. Then, the path of steepest ascent and the Box-Behnken design were adopted for further optimization, and the optimum concentration levels and the relationships among these factors was found out by quadratic regression model equation with Design-Expert statistic methods, the optimal concentration of the variables were determined as: glucose 37.22 g/L, yeast extract 6.9 g/L, (NH4)2SO4 6.55 g/L. Under such conditions, the concentration of ε-PL was increased to 8.11 g/L, which was 24.6% higher than the maximum value in the single factor tests.