Chronic otitis media or tympanic membrane (TM) perforation is one of the most common otologic diseases. Surgical tympanoplasty remains the best treatment option despite the fact that paper patches are frequently used. Although paper patches are not biocompatible or effective, tympanoplasty is an expensive, complex surgery. Tissue engineering techniques offer a new treatment strategy for TM regeneration. In this study, novel tissue-engineered artificial eardrums were fabricated from water-soluble chitosan, which is known to be a good wound-healing biomaterial. The characteristics, cytotoxicity, and healing effects of several water-soluble chitosan patches (WSCPs) made using various concentrations of water-soluble chitosan and glycerol were investigated. The optimal WSCP was fabricated with 3% water-soluble chitosan and 3% glycerol, and it had a thickness of about 35 mum, a tensile strength of 7 MPa, a percent elongation of 101%, a hydrophilic surface, and no cytotoxicity. In vivo studies showed that the WSCPs were more effective than spontaneous healing for the repair of traumatic TM perforations. The healed TMs to which WSCPs were applied had a much higher density of collagen fibers and a better lamina propria layer structure than spontaneously healed TMs.