Streptomyces albulus UN2?71 was selected as original strain and its protoplast was treated by diethyl sulfate (DES) to obtain high ε-polylysine producing mutants. Through primary screening and confirmed screening with shake flask cultures, one strain designated as D3-32 with a good genetic stability has been acquired, whose production in the shake flask reached 1.56 g/L, which was about 49.43% higher than that of original strain. In the fermentation test with 2.3 L fermentor, after a pH-controlled two-phase fermentation, the highest production of ε-polylysine reached 4.59 g/L, which was increased by 2.65 times compared to that of the original strain.