Abstract: A thermo-separating aqueous two-phase system composed of Dehypon® LS 54, a polymeric surfactant and the waxy maize starch (amylopectin starch), has been used for partitioning of cutinase as a model protein. The phase diagram obtained for this novel polymer-polymer two-phase system shows two-phases with high polymer concentration. The waxy maize starch is enriched in the bottom phase while the copolymer of Ethylene Oxide (EO) and Propylene Oxide (PO) is found in the upper phase. Since, this copolymer (Dehypon® LS 54) is the thermo-reactive, the upper phase can be removed and heated above the copolymer’s cloud-point resulting in the formation of a new two-phase system with a lower water phase containing the target protein and an upper copolymer-rich phase. Present results show that the systems were formed by the waxy maize starch and a copolymer Dehypon® LS 54 may be considered as an interesting alternative to be used in protein purification due to their low cost, in addition, they offer a viable solution to problems of polymer removal and recycling.