The content of free primary amine in the solution was determined by spectrophotometric assay according to that ο-phthaladehyde (OPA) reagent and primary amine could occur fluorescence reaction. The effect of dosage, reaction temperature, time and pH of transglutaminase (TGase) treatment on the free primary amine of soy protein isolate (SPI) was investigated with this method, and the influencial factors were optimized by response surface method. The results showed that the optimal reaction condition was TGase dosage 20 U/g, temperature 52 ℃, time 60 min and pH 7. After TGase-catalyzed modification, the free amine content of SPI were reduced from 0.059 μmol to 0.028 μmol.