The phase equilibrium and protein partitioning in the aqueous micellar two-phase system composed of nonionic surfactant polyoxyethylene octyl phenyl ether (Triton X-100) and polymer polyethylene glycol (PEG) with different low molecular weights are studied. The phase diagrams under various PEG molecular weight, salt concentration and pH value are determined at 298 K. The results show that the biphase areas of the aqueous micellar two-phase system are expanded with an increase in PEG molecular weight or salt concentration, while the biphase areas and binodal curves are nearly unchanged by varying pH values. With raising PEG molecular weight, the tie lines lengths increase, and the tie lines slopes decrease first and then increase. Using lysozyme and bovine serum albumin (BSA) as model proteins, protein partitioning in the aqueous micellar two-phase system is investigated. As the hydrophilic proteins, both lysozyme and BSA partition preferably to the PEG-rich phases. The partition coefficients of proteins increase with increasing tie line length, or with decreasing PEG molecular weight and protein molecular size. These results indicate that the excluded volume interactions play an important role in protein partitioning in the aqueous micellar two-phase system composed of surfactant and polymer. Crown Copyright (C) 2012 Published by Elsevier B.V. All rights reserved.