Empirical modeling the partition behavior and recovery of a recombinant Pseudomonas putida POS-F84 proline dehydrogenase (ProDH) in aqueous two-phase systems (ATPS) was carried out by response surface methodology (RSM). Polyethylene glycol 1000 (PEG-1000) concentration, sodium carbonate concentration, and pH, which were the most important factors, were chosen for modeling the partition feature of enzyme. The adequacy of the models was investigated by means of variance analysis. Also, to confirm the efficiency of the ATPS in partition and purification of recombinant ProDH, purity and enzymatic activity was studied. After numerical optimization, an optimal ATPS composed of 14.33% PEG-1000 and 11.79% sodium carbonate at pH 7.48 was achieved. Yield, purification factor, and recovery were 81.41%, 60.82, and 270.82%, respectively. Purified recombinant ProDH was found as a single protein band into the upper PEG-rich phase and the specific activity was calculated to be 46.23 +/- 2.1 U mg. Collectively, our data showed that the RSM could be an appropriate and powerful tool to define the best ATPS system for recovery and purification of P. putida ProDH.