The recyclable aqueous two-phase systems (ATPS) responding to environmental stimuli have been widely studied in the purification of biologics. In this study, a thermo-responsive polymer P(NE)was copolymerized after optimization of monomer ratio. In addition, its lower critical solution temperature (LCST, 31 degrees C) and first recovery (99.43%) were determined. Then, P(NE)was used to form two recyclable ATPS with another thermo-responsive polymer P(NDBN)and a pH-responsive polymer P-ADB4.91, which were polymers already prepared in the lab. Meanwhile, the partition behavior of microbial transglutaminase (MTG) was explored using these two ATPS. The result showed that the P-NE/P(ADB4.91)ATPS was superior to P-NDBN/P(NE)ATPS owing to its faster phase formation and better partition performance. In order to optimize the partition behavior, several parameters were investigated based on P-NE/P(ADB4.91)ATPS. In the ATPS constructed with 2.5% (w/v) P(NE)and 3.5% (w/v) P-ADB4.91, the maximal partition coefficient (1/K-E) and enzymatic recovery (ERB) of MTG were 12.9 and 95.21% in the presence of 10 mM KCl when the temperature, pH, and the addition amount of MTG were 25 degrees C, 7.0, and 2 mg/mL, respectively.