The separation of L-lysine in aqueous two-phase system containing polyethylene glycol( PEG) and sodium sulfate was carried out based on the physicochemical properties of L-lysine. According to the extraction yield and partition coefficient,the influences of mass concentration of the compositions,pH value,temperature and their interactions on L-lysine extraction were examined by single-factor and orthogonal experiments. The results showed that L-lysine preferred to partition in the bottom salt-rich phase. The content of two components,pH value,as well as the interaction of Na2SO4content and pH value had significant influence on the extraction. The optimum extraction conditions were 20%( mass) of polyethylene glycol with molecular weight 1000 and 14%( mass) of Na2SO4at pH 5. 5 and 30℃. Under the optimal conditions,the average yield and the partition coefficient of L-lysine were 96. 5% and 0. 033 respectively using 1. 0 mg / mL L-lysine solution. And a preliminary separation from simulated fermentation broth with the same concentration of L-lysine displayed a extraction yield and partition coefficient were 94. 8% and 0. 057,respectively.