The technique of three-phase partitioning (TPP) was used to purify a bifunctional amylase/protease inhibitor from ragi (Eleusine coracana). This process of purification is a potential method used for separation of proteins directly from large volumes of crude suspension. It involves the addition of a salt (ammonium sulphate) to the crude extract followed by the addition of an organic solvent (t-butanol). The addition of t-butanol, in the presence of ammonium sulphate pushes the protein out of the solution to form an interfacial precipitate layer between the lower aqueous and upper organic layers. The process was carried out in two steps. The various conditions required for attaining efficient purification of the protein fractions were optimized. It was seen that 30% ammonium sulphate saturation with 1:1 ratio of crude extract to tert-butanol gave 8.9- and 8.65-fold purification with 83% and 80% yield of amylase inhibitor and trypsin inhibitor, respectively, in step I. In TPP-step II, 60% ammonium sulphate saturation and ratio of aqueous phase to t-butanol of 1:2 gave maximum 20.1- and 16-fold purification with 39.5% and 32% yield of amylase inhibitor and trypsin inhibitor, respectively. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the inhibitor protein showed substantial purification and the molecular weight of the protein was found to be 14 kDa. (c) 2006 Elsevier Ltd. All rights reserved.