Purification of a desired protein from fermentation broth containing a wide variety of biomolecules is one of the major challenges in the biotechnology industry. One possible strategy for this challenge, an aqueous two-phase extraction (APTE) can be used as the primary downstream processing for partial purification of industrial enzymes. In the present study, the feasibility of utilizing ATPE for the partitioning behavior of P-galactosidase (from Aspergilus niger) was investigated. The results showed that the PEG2000/(NH4)(2)HPO4 system was suitable for the extraction of P-galactosidase from cell free fermentation broth of A. niger PTCC 1050. In the following, multilevel factorial design was used to evaluate the effects of three independent variables (PEG to salt weight ratio, fermentation broth loaded mass and system pH) on p-galactosidase extraction. The results indicated that PEG to salt weight ratio and fermentation broth loaded mass was the major contributory factors for P-galactosidase partitioning. Among the different conditions studied, a phase system having a composition of 15.24% (w/w) PEG2000 and 17.14% (w/w) (NH4)(2)HPO4 at pH 7 with loaded fermentation broth of 5% (w/w) gave the best overall results in term of purification degree. (C) 2014 Elsevier B.V. All rights reserved.