In the current investigation, aqueous two-phase methodology with polyethylene glycol (PEG) 6000/phosphate salt has been used for single-step purification of beta-galactosidase from recombinant Pichia pastoris. Optimized parameters with 12% (w/w) salt concentration, 25% (w/w) polymer concentration, 0.6 (mg/ml) protein load and 0.1 M ionic concentration resulted in a maximum of 4.7 purification fold with a 97% yield. The enzyme kinetic study of purified protein revealed V-m and K-m of 97.087 (U/mg), 0.027(U/mg), 0.07 (U/mg) and 0.7 (mM), 11.13 (mM), 10.73 (mM) with O-nitrophenyl-beta-D-galactopyranoside, lactose and milk substrate, respectively.