Abstract: Aqueous two-phase systems (ATPSs) are formed by 2 materials with different structures, such as polyethylene glycol (PEG) and dextran or a polymer and a salt. Possessing many important advantages such as short processing time, low energy consumption, relative reliability in scale-up and biocompatibility in environment, ATPSs are widely used for separation and purification of various proteins and nucleic acids. Based on this theory, the metal affinity aqueous two-phase systems have been reported which introduced the metal ion to PEG to enhance the efficiency of the extraction. Papain (EC3.4.22.2) is an endolytic cysteine protease obtained from the latex of papaya (Carica papaya), which can catalyze the hydrolysis of peptide bonds composed by basic amino acids. As its wide pH span for optimum activity, high sensitivity, temperature stability, low price and short response time, papain has been extensively used in medicine, cell, detergents, leather, textiles, cosmetic and food fields, and played an important role in our daily life. With the development of purification technology, more and more novel ATPSs have been used for the extraction of papain. However, most researchers have focused on the optimization of partitioning conditions and scarcely concentrated on the papain's partition model, especially the partition model in affinity aqueous two-phase systems. Overall, the papain partition behavior in ATPSs is affected by the type and concentration of polymer and salt, the environment temperature, the pH value and the substance for separation. The effect of pH value on papain activity and the effect of PEG-IDA-Fe3+ on enzyme activity were investigated in this paper. Results showed that the pH value of 4.0-9.0 had little effect on enzyme activity and the papain activity could reach the highest (3478 U/mg) at the pH value of 7.0, while PEG-IDA-Fe3+ would restrain papain activity slightly. To ensure the partition behavior of papain in PEG/PEG-IDA-Fe3+/(NH4)2SO4 affinity ATPS was selective and predictable, the triangular phase diagrams of ATPSs were measured by means of cloud point under 298.15 K. Two kinds of triangular phase diagrams showed that 10% PEG-IDA-Fe3+ replacement had little effect on the phase diagram. Experiments can be conducted according to the 2 kinds of phase diagrams. Based on the correlation between the logarithm of the partition coefficient and the concentration difference of each component, the partition model of papain in PEG/(NH4)2SO4 ATPS was established. The mean relative deviation of the equation model was 7.02% which was less than other similar models. In addition, the impact factor was introduced to PEG/PEG-IDA-Fe3+/(NH4)2SO4 system to establish affinity partition model of papain. The relative errors between the experimental and predicted values of the partition coefficient were less than 15%, which could represent the affinity partition behavior of papain in PEG/PEG-IDA-Fe3+/(NH4)2SO4 system. The results in this paper indicate that the new model is potential for predicting papain's partition coefficient in metal chelated ATPSs.