In this study, a pH shock strategy was employed to enhance epsilon-poly-L-lysine (epsilon-PL) production from glucose. In the conventional fermentation, in the early stage, only 13% of total epsilon-PL production is achieved in 25% of the entire fermentation period, which severely affected e-PL productivity. To improve the efficiency of epsilon-PL production during fermentation, a novel two-stage fermentation, namely culture and fermentation stages, was proposed on the basis of the analysis of conventional pH shock fermentation. After optimization of parameters such as inoculum growth conditions, initial fermentation pH, and inoculum volume, the epsilon-PL production and productivity achieved using the novel fermentation process in a 5-L fermenter reached 32.22 g/L and 5.86 g/L/day, which were 32.3% and 36.6% higher, respectively, when compared with those obtained in conventional fermentation. Furthermore, evaluation of acid tolerance of mycelia collected from the pH shock fermentation showed that pH shock enhanced epsilon-PL production, which might be related to the acid tolerance of Streptomyces albulus and pH stress (pH 3.0). The results obtained could be useful for large-scale epsilon-PL production and to provide new information on epsilon-PL biosynthesis mechanism.