Abstract: Aqueous two-phase system was used to separate alkaline xylanase from recombinant Pichia pastoris.The effects of molecular weight of the PEG, PEG concentration, phase forming salts, salt concentration, neutral salt (Na Cl) concentration and p H on enzyme partitioning and purification were investigated. The results showed that PEG 4000 concentration 20%, disodium hydrogen phosphate concentration 18%, without Na Cl, under the condition of p H6.0 were the optimal conditions. And the best results were achieved as follows:partition coefficient (K) 8.51, enzyme activity recovery (Y) 87.2% and the purification factor (PF) 2.54.