The aim of this research is to study the technology of aqueous two-phase extraction of nisin. Several important parameters affecting the extraction efficiency were studied and optimized, including different molecular weights of PEG, the concentration of PEG, different minerals, the concentration of the salt and pH. Furthermore, Box-Behnken central composite utilizing 3 factors with 3 levels was applied to optimize the isolation and depuration process of nisin. Response surface analysis method was applied to determine the effect of casual factors on extraction efficiency of nisin. The results showed that the optimum aqueous two-phase extractions in dependent variables for separating nisin were as follow: 18.6% PEG 4000, 11.7% NaH2PO4, pH 2.6. Under the optimized conditions, the extraction rate of nisin was about 84.3%.