Microbial transglutaminase (MTG) has been widely used in the food and pharmaceuticals industries. In this study, MTG was purified using affinity precipitation with an affinity polymer (P-MMDN-T), which was synthesized using a pH-responsive polymer (PMMDN) coupled with L-thyroxin as an affinity ligand. Interactions between MTG and P-MMDN-T were investigated using turbidimetric titration, zeta potential measurements, and low-field nuclear magnetic resonance (LF-NMR). We found different behaviors, architectures, and phase states of pH-dependent interactions between MTG and P-MMDN-T interactions. Binding energetics between MTG and P-MMDN-T were determined by isothermal titration calorimetry (ITC). The isoelectric point (pI) of the affinity polymer was 4.65 and was recovered with 96.7% efficiency after recycling the polymer three times. The optimal adsorption condition was 0.02 mol/L phosphate buffer (pH 6.0) with 1.0 mol/L NaCl at 30.0A degrees C and a ligand density of 50.0 mu mol/g. The maximum elution recoveries of total MTG were 98.44% (protein) with 92.19% (activity) using 0.02 mol/L pH 10.0 Gly-NaOH as the eluent.