In this work, partitioning of α-amylase and Bovine Serum Albumin (BSA) in aqueous two-phase systems was investigated. The two-phase aqueous systems contained polyethylene glycol (PEG) and phosphate salts. Results, show bi-nodal curves for the phases at equilibrium. The effects of molecular weight and concentration of PEG, phosphate salts, pH and presence of sodium chloride on partition coefficients of the α-amylase and BSA were investigated. The results show that by increasing the molecular weight of PEG from 2000 to 15000, the partitioning tendency of α-amylase to the top phase (or light phase, rich in PEG) nearly doubles. Also, on increasing the weight ratio of phosphate to PEG, and in the presence of sodium chloride, the partition coefficient of both α-amylase and BSA show an increase. When the weight ratio of phosphate to PEG was increased from 0.48 to 3.73 for the system containing PEG 15000 and NaH2PO4, the partition coefficient of α-amylase was increased from 0.76 to 1.44. Whereas, for the system containing NaH2PO4 and PEG 2000, on addition of 0.25 molar sodium chloride, the α-amylase partition coefficient increases 6 times. The effect of pH on the partition coefficient of α-amylase depends on the type of system. The results show that in the case of BSA the partition coefficient is higher for higher molecular weights of PEG and also for higher phosphate to PEG ratio. By decreasing the PEG molecular weight from 15000 to 2000, the partition coefficient increases 3 times, depending on the weight percent of phosphate salts. However, for systems containing BSA, addition of NaCl to the systems can cause either an increase or decrease in the partition coefficient of BSA.