In clinical experience, tympanic membrane(TM) perforation has many causes and various progressions of the healing process. Local application of some drug without treatment delay may improve the wound's healing. Hyaluronic acid in an extracellular matrix has the property of healing the wound. This study was performed in order to evaluate the effect of local application of hyaluronic acids(HA) in ear drum perforation. The following results were obtained : 1) The average closing time of TM perforations was 4.6 days in the HA group and 9.7 days in the control group. 2) The arrangement of collagen fibers in the stroma was well organized to show parallel pattern to the surface of the TM in the HA group, but the collagen fibers of the control group showed irregular arrangement at five days after the perforation. 3) The closure of the perforation by the keratinizing epithelium of the TM was better in the HA group than in the control group at five days after the perforation. The cell organelles of the epithelium in the HA group were well developed. 4) At 20 days after the perforation, the collagen fibers of the control group showed irregular arrangement although they were plentiful. In the HA group, the epithelium was well differentiated to show well developed desmosomes and tonofilaments. The stroma of the HA group showed well organized collagen fibers. This study concluded that HA has a good effect in the healing of the TM perforation which includes fast recovery from the trauma, well organized collagen fibers in the stroma and well differentiated cell and cell organelles.