The invention discloses a preparation method of an animal model with oral ulcer caused by fire excess from yin deficiency. The method comprises selecting healthy rats, carrying out subcutaneous injection on the rats with a triiodothyronine solution according to the dosage of 5-200 micrograms per kilogram for 14-21 days, carrying out impregnation on a cotton ball with phenol, and firing the rat lateral buccal mucosa to form a white wound so that a desired animal model can be obtained after 24-48h. Compared with the prior art, the method is used for preparation of an animal model with oral ulcercaused by fire excess from yin deficiency. The model can induce the oral ulcer based on the fire excess from yin deficiency according to the traditional Chinese medicine differentiation of symptoms and signs, well satisfies clinical practice, has high feasibility and high applicability and has a great application value in heat and fire removal, tissue damage reduction and ulcer healing promotion.