In this paper, gelatin-based films were prepared by mixing gelatin with glycerol, and microbial transglutaminase (mTG) which was used as cross linking agent. The result of SEM confirmed that a dense network structure was formed after mTG treatment comparing to the native. The effect of mTG on water barrier property of films was investigated by measuring the water vapor permeability (WVP). The results showed that the concentration of gelatin has a significant influence on the water barrier property of gelatin-based film which was treated with mTG. The WVP value of the films decreased sharply after enzymatic cross-linking when gelatin concentration increased to 8%, while no evident change of the WVP can be found when the gelatin concentrations was lower than 8%.