Three phase partitioning (TPP), an efficient bioseparation technique, was used to purify nattokinase from fermentation broth of Bacillus natto NRRL-3666. TPP is a relatively recent technique that uses combination of ammonium sulphate and t-butanol to precipitate protein from crude extracts. The precipitated protein forms interface between the lower aqueous layer and the upper organic layer. The key parameters that were studied for efficient purification were temperature, pH, concentration of ammonium sulphate and t-butanol. The pH of 8, temperature of 37 degrees C, ammonium sulphate 30% (w/v) and ratio of crude extract to t-butanol 1:1.5 gave the most desired results. A single stage TPP led to 5.6-fold purification with an activity recovery of 129.5%. Nattokinase was freeze dried by using different excipients. There was no effect on the secondary structure of nattokinase while using t-butanol (co-solvent) for freeze drying. But there was reduction in drying time as well as the nattokinase cake became more porous. (C) 2014 Elsevier B.V. All rights reserved.