The optimal conditions for propionic acid extraction were determined by response surface methodology in an aqueous two-phase system. A second-order central composite design was applied to evaluate the effects of four independent variables (PEG 4000 concentration (%w/w), ammonium sulfate concentration (%w/w), temperature (degrees C), and propionic acid concentration (%w/w)) on propionic acid extraction yield. Correlation analysis of the mathematical regression model indicated that the quadratic polynomial model could be employed to optimize the extraction. The optimal conditions were 30.71 (%w/w), 31.77 (%w/w) 37.37 (degrees C), and 0.72 (%w/w) for PEG 4000 concentration, ammonium sulfate concentration, temperature, and propionic acid concentration respectively. Maximum yield of extracted propionic acid from the experiments was determined to be 69.78% under the optimal conditions.