A method for measuring the integrity of intestinal epithelial tight injunction and application thereof are disclosed. The method measures intestinal epithelial impedance of animal intestine in-vitro and reflects damage of intestinal epithelial tight junction in intestine part according to the decrease of intestinal epithelial impedance. The method operates easily and needs small quantity of samples, capable of reflecting integrity of different part of intestinal epithelial junction without the influence of the blood flow and metabolism. The method can be used in animal experimental research in intestine barrier disorder.