The use of thermosensitive polymers in an aqueous two-phase system was studied. Poly(N-isopropylacrylamide) (PNIPAAm) and poly(N-vinylisobutyramide) (PNVIBA) were used as thermosensitive polymers. Both polymers could form aqueous two-phase with dextran, respectively. The phase diagrams of each system were successfully obtained. Using myoglobin as a model protein, a preliminary separation study was performed. The separation ability of both polymers was higher than that from the poly(ethylene glycol)-dextran system. Protein separation ability appeared to be related to the hydrophilic/hydrophobic balance of the polymers. Both PNVIBA and PNIPAAm rich phases maintained their thermosensitivity after two-phase formation. PNVIBA and PNIPAAm are useful as polymers for a functional aqueous two-phase system.