To develop a polyethylene glycol (PEG)-potassium phosphate aqueous two-phase system for extraction of a alkaline protease and the determine suitable conditions for phase forming and alkaline protease extraction by aqueous two-phase system. The partitioning of an alkaline protease from Bacillus subtilis NS 99 in aqueous two-phase systems of polyethylene glycol with MW 1000 Da and potassium phosphate salts was examined at ambient temperature (30+-2ํC) and atmospheric pressure. The top phase was rich in PEG while the bottom phase was rich in phosphate. Factors such as pH, concentration of sodium chloride (NaCl) and tie-line length were found to influence phase diagram and partition coefficient (K) in range of initial concentration of PEG1000, 5-70% (w/w); pH at 7.5, 8.5, 9.5 and 10.5, and NaCl concentration at 0, 4, 7 and 10% (w/w). In the forming of aqueous two-phase, pH was found to have very slight or almost no effect. In contrast, increase in NaCl concentration was discovered to reduce PEG and/or potassium phosphate concentrations used in phase formation. The alkaline protease partition coefficient was enhanced with increasing pH but decreased with increasing tie-line length and concentration of NaCl. The suitable conditions found for alkaline protease extraction were 18.00% (w/w) PEG1000, 13.01% (w/w) potassium phosphate and 0% (w/w) NaCl at pH 9.5, ambient temperature (30+-2ํC) and atmospheric pressure which gave the K value of 20.0 and percent yield of 95.1.