The increasing demand for safe and effective methods for the separation of biological materials combining the advantageous properties of polyethylene glycol (PEG) (1500 g mol −1 or 4000 g mol −1) and ionic liquid has led to attractive and promising applications of the new aqueous biphasic systems. In this work, the liquid-liquid extraction process with aqueous two-phase systems (ATPS) was studied for α-amylase partitioning. The phase diagrams composed of PEG (1500 or 4000) g mol −1 + 1-ethyl-3-methylimidazolium chloride + water, were determined experimentally by the turbidimetric method at different temperatures T = (293.15, 303.15, 313.15 and 323.15) K. The effect of temperature and polymer molar mass on the phase diagrams, as well as the phase densities, refractive indexes and electrical conductivity were evaluated. Subsequently, the α-amylase partitioning was performed, where the partition coefficient was determined, besides the activity and selectivity. It was found that a temperature decrease and a polymer molar mass increase favored the formation of two phases. It was also found that α-amylase showed a preference for the bottom phase composed of PEG and the systems composed of PEG 1500 presented better results. Keyword Liquid-liquid extraction • Enzyme • Partition • Amylase Abbreviations ATPS Aqueous two-phase systems PEG Polyethylene glycol [C 2 min]Cl 1-Ethyl-3-methylimidazolium chloride TLL Tie-line lengths STL Tie-line slope U One enzyme activity unit releases 1 mmol of reducing sugar per mL of extract per minute, U mL −1 ART Total reducing sugars produced in the hydrolysis step, mg mL −1 VT Total volume used in the hydrolysis, mL V c Volume of sample used in the hydrolysis, mL R t Reaction time, min BSA Bovine serum albumin K p Protein partition coefficient C t Equilibrium concentration of the total protein in the top, mg mL −1 C b Equilibrium concentration of the total protein in the bottom, mg mL −1 K e Partition coefficient for enzyme activity A t Enzyme activity in the top, mg mL −1 A b Enzyme activity in the bottom, mg mL −1 Δ tr G Gibbs free energy variation, kJ mol −1 T Absolute temperature, K R Ideal gas constant, kJ mol −1 K −1