The invention discloses an animal intestinal mucosa extract and a preparation method as well as application thereof. The animal intestinal mucosa extract is prepared by the method comprising the following steps: (1) taking small intestine of an in vitro animal, scraping the mucosa layer on the intestinal wall, centrifuging and collecting precipitates; (2) adding the precipitates into water, then adding trypsin into the redistilled water for carrying out enzymolysis I, and after the enzymolysis is finished, inactivating enzymes and cooling; then, adding papain into the redistilled water for carrying out enzymolysis II, and after the enzymolysis is finished, inactivating enzymes and cooling; finally, centrifuging and taking the supernatant solution to obtain an intestinal mucosa enzymolysis solution; and (3) adding an acetic acid solution of which the mass concentration is 5%-10% into the intestinal mucosa enzymolysis solution for stirring and leaching, then centrifuging the leaching solution, and collecting the supernatant solution, and regulating the pH value of the supernatant solution to be 6.0-7.0, freezing and drying to obtain the animal intestinal mucosa extract. The test proves that the intestinal mucosa extract has antibacterial activity to bacteria (such as Escherichia coli).