An integrated fermentation-separation process for the production of ε-poly-L-lysine (ε-PL) was investigated. Through the screening of several aqueous two-phase systems, the extraction of ε -PL by ethanol/ammonium sulfate aqueous two-phase system was investigated in detail. The results indicated that the highest partition coefficient (18.33) and recovery (98.02%) of ε-PL were obtained with an aqueous two-phase system composed of 20% (w/w) ethanol and 20% (w/w) ammonium sulfate, pH9.5. The effect of multi-stage extraction was investigated. It was found that the effect of extraction for three times was the best, the removal rate of protein and pigment were 85.66% and 60.00%, respectively, and the yield of ε -PL was 96.15%. Finally, the top phase after three times of extraction was desalted and concentrated by ultrafiltration membrane, and the yield and purity of the product reached 68.28% and 92.39%, respectively.