Abstract: The papain was separated and extracted by PEG/Na2SO4 aqueous two-phase system in this study.The influence of reagent and pH value on activity of papain were investigated, in the meantime, the influence of PEG with different molecular weights, the mass fraction of Na2SO4 and PEG, pH and enzyme added on distribution behavior in aqueous two-phase system was discussed. The separation condition of papain in aqueous two-phase system was optimizated by response surface method, and amplified in laboratory scale.The study showed that:the effect of Na2SO4 mass fraction on the distribution behavior was significant, followed by PEG mass fraction, the effect of pH on the distribution behavior was not significant. The partition coefficient and recovery rate of papain in aqueous two-phase system achieved 3.1 and 82.6% under the condition of molecular weights of PEG6000, mass fraction of PEG 16.0%, mass fraction of Na2SO418.0%, pH7.0, enzyme added 1.0mg/g. The results showed that:the optimization on extraction of papain by aqueous two-phase system was feasible and could be used to predict the actual.