Abstract: To investigate the effect of transglutaminase(TGase)on the performance and microstructure of whey protein concentrate(WPC)-nanocrystalline cellulose(NCC)composite film,WPC and NCC were used as raw materials,mechanical properties and barrier properties of WPC-NCC composite films with TGase treatment were optimized and the effect of cross-linking of TGase on the secondary structure of protein molecule and the microstructure of the composite film were investigated in our study. The results showed that tensile strength of WPC-NCC composite film reached to 2.25 MPa,elongation at break reached to 86.75%,and water vapor permeability decreased to 4.40×10^-12 g mPa^-1s^-1m^-2 when the amount of TGase added at 12 U/g protein. TGase effectively improved the mechanical properties and water vapor barrier properties of WPC-NCC composite film. TGase promoted protein structure to change in a stable and orderly direction,reduced the number of pores and pore size in the composite film,and made the microstructure of film denser. It also showed that the improvement of mechanical properties and water vapor barrier property were closely related to the microstructure.