PEG-Dextran and PEG-salt aqueous two-phase systems (ATPS) have been applied to separate glutathione (GSH) from crude yeast extracts. Single-factor experiments were carried out to determine the important factors influencing the partition coefficient and extraction yield. The effect of PEG molecular weight, phase-forming components, PEG and Dextran concentration, pH value, and temperature on the GSH partitioning behavior in ATPS was investigated. Three factors, Dextran concentration, pH value, and temperature, were confirmed to have significant influence on the partition coefficient and extraction yield. These factors were further analyzed with the aid of central composite rotatable design and response surface methodology. The optimal conditions for GSH extraction in the PEGDextran system were determined, including PEG molecular weight 6,000, 10% PEG concentration, 14% Dextran concentration, pH 5.2, and temperature 32 ℃. A high extraction yield (83.55%) of GSH from crude yeast extracts was achieved under these optimized conditions. This work is very helpful for developing one efficient and cost-effective process for the separation and purification of GSH from yeast broths.