The invention discloses a purification method of an MTGase (Microbial Transglutaminase) crude enzyme. The purification method comprises the following steps: dissolving MTGase crude enzyme powder with water to prepare an MTGase crude enzyme solution with the concentration of 1 weight percent to 2 weight percent; preparing a PEG (Polyethylene Glycol)/(NH4)2SO4 double-water-phase solution to form an upper-layer PEG phase and a lower-layer salt phase; uniformly mixing the MTGase crude enzyme solution with the PEG/(NH4)2SO4 double-water-phase solution; standing at 20 DEG C to 40 DEG C for 1h to 2h; enabling MTGase to enter the salt phase in a double-water-phase system and collecting the salt phase in the double-water-phase system; carrying out nanofiltration membrane treatment on the collected salt phase and collecting inter-membrane liquid of the nanofiltration membrane treatment, so as to obtain purified MTGase. The purification method has the advantages that purification is simple and convenient, the purity of the MTGase obtained by purification and separation is relatively high and the recycling rate is high.