To investigate the effect of glycine on ε-poly-L-lysine (ε-PL) production of Streptomyces diastatochromogenes Cbγ4, certain concentrations of glycine were added in the seed culture. When 3g/L glycine was added at the early stage in seed culture, the accumulation of ε-PL reached 0.51g/L, which was increased by 27.5% than that of the control group, and the aspartokinase activity was improved obviously. In 5-L jar fermentation, the biomass and the consume rate of glucose were greatly enhanced by adding 3g/L glycine in seed culture, and the ε-PL yield was increased to 12.87g/L, meanwhile, the conversion rate of carbon resource to ε-PL was 30.6% higher than the control group. Therefore, glycine could be used as a novel nutrient for ε-PL production.