A new aqueous two-phase system composed of a non-ionic surfactant, Dehypon (R) LS54 (abbreviated as Ls54) and a starch derivative, K4484 (R) Dextrin (abbreviated as Dx), was prepared in different compositions and the formed phases were characterized for physical properties such as density and viscosity. The system was later investigated to be applied for the extraction cutinase enzyme. The phase formation behaviour subjected to temperature and pH were studied. The system equilibrium represented by a binodal diagram, was constructed by using cloud point method. The binodal curve was suitably fitted with Merchuk equation. The correlated tie lines associated with the equation were determined by the use of lever arm rule of the mass phase ratio. The results showed that the tie line length (TLL) had a strong influence on system's properties such as phase volume ratio and viscosity difference, however, the density of systems were not strongly affected. Unlike pH, the temperature changes clearly affected the phase formation ratio. Furthermore, cutinase incubation in the Ls54 and Dx solutions had resulted small variation in cutinase activity, suggesting that the presence of the phase-forming polymer did not reduce the stability of the targeted enzyme. The effects of pH, tie line length and phase volume ratio on partition coefficient, recovery and purification of the enzyme were further investigated. The optimal pH of system containing 22%w/w Ls54 and 12.5%w/w Dx was found at pH 8.0 with the partition coefficient, k(cut) 1.32, yield 65% and purification factor of 6.92. (C) 2014 Elsevier B.V. All rights reserved.