A microbial transglutaminase (MTG) was efficiently purified by using pH-responsive affinity precipitation with Crocein orange G (COG) as the affinity ligand. The docking method was used to identify the appropriate ligand. The molecular simulation results were compared with the label-free detection data analyzed by ForteBio's Octet. A pH-responsive polymer, P-MMDN, was polymerized and subsequently coupled with COG as the ligand. The isoelectric point (pI) and recovery of P-MMDN and P-MMDN-COG were 4.51, 99.8,% and 4.78, 98.1 %, respectively. The optimal adsorption conditions were found to be a ligand density of 60.0 mu mol/g, pH 7.0, and 0.2 mol/L NaCl. The adsorption isotherm showed that the maximum adsorption capacity was 91.32 mg/g polymer and the dissociation constant was 0.021 mg/mL. Interaction information between P-MMDN-COG and MTG in the whole process of affinity precipitation were obtained by Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM). PB (0.02 mol/L pH 7.0) in 20.0 % glycol was used to elute the binding MTG from P-MMDN-COG. Under these conditions, electrophoretically pure MTG was obtained in only one step with elution recoveries of 98.96 % (protein) and 95.09 % (activity).