Growing applications of phenylalanine dehydrogenase (PheDH) enzyme in the medical and pharmaceutical industries encourages researchers to seek simple, fast and economical alternative purification methods. With goal of finding a new technique, the extraction and purification of recombinant Bacillus badius PheDH in polyethylene glycol 6000 (PEG-6000) and ammonium sulfate aqueous two-phase systems (ATPS) was studied. Investigation on the effects of PEG molecular weight (MW) and concentration, (NH4)2SO4 concentration, pH, temperature and NaCl addition on partition behavior was also performed. The optimized system was 8.5% (w/w) PEG-6000, 17.5% (w/w) (NH4)2SO4 and 13% (w/w) NaCl at pH 8.0. The specific activity, yield, purification factor, recovery and partition coefficient were obtained 10424.97 U/mg, 95.85%, 474.3, 141% and 92.57, respectively. Briefly, we described the application of ATPS as an interesting and powerful technique for purifying and recovery of recombinant PheDH.