An unconventional aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) and sodium carbonate was developed and optimized by employing response surface methodology for separation of Rhizopus niveus lipase. A five-level central composite design was applied to evaluate the optimal level of three process variables in order to obtain maximum lipase separation. Experimental data were analyzed by regression and a polynomial model was created which was found significant. The maximum partition coefficient was achieved with the system PEG 4000/sodium carbonate. Validation experiments confirmed the high accordance of predicted and experimental results. The optimized ATPS can be applied as a suitable cost-effective system for lipase extraction.