ε-poly-L-lysine (ε-PL) is a natural homo-polymer of microbial origin, consisting of 25~35 L-lysine monomers, which is mainly produced by aerobic microbial fermentation and secreted to extracellular accumulation. Due to its wide antimicrobial spectrum and high safety, ε-PL has been successfully used as a food preservative. Besides, as a safe and green biopolymer, it has also been extensively applied in biomedical, chemical, and many other fields. Unfortunately, the efficient microbial production of ε-PL has reached a bottleneck owing to the limitations of low productivity, long fermentation period and unstable fermentation process, which can not satisfy the demand of industrialized production and brought obstacles to its popularity. To tackle these issues, S. albulus FMME-545RX with high tolerance to rifamycin was firstly screened and obtained by atmospheric and room temperature plasmas (ARTP) mutagenesis combined with ribosomal engineering, which could produce 2.44 g/L of ε-PL, with an increase of 105% in comparison with that of the parent strain S. albulus FMME-545. Then, a series of fermentation optimization strategies, including carbon sources regulation, pH control and dissolved oxygen (DO) regulation, were employed in increasing the production of ε-PL. The final results demonstrated that the mixed carbon source of glucose and sucrose fermentation was helpful to improve the metabolic intensity of bacteria; the addition of sodium citrate in the fermentation process can effectively improve the ability of the bacteria to resist the acidic environment; the optimum pH and DO values for product synthesis were 3.80 and 30%, respectively. Finally, under the controlled fed-batch fermentation, the production, productivity, and dry cell weight (DCW) of ε-PL reached up to 53.0 g/L, 6.63 g/(L?d), and 0.88 g/g, respectively, which were 130%, 131%, and 118% higher than those of the parent strain S. albulus FMME-545. Taken together, this study shows great potential for industrial production of ε-PL and the strategies described here also pave the way to the production of other value-added chemicals.