Objectives Chronic otitis media is a long-term infection of the middle ear. It is characterized by persistent discharge from the middle ear through a perforated tympanic membrane. It is one of the most common causes of preventable hearing loss, especially in developing countries. Precise estimation of the size of tympanic membrane perforation is essential for successful clinical management. In this study, we developed a smartphone-based application to calculate the ratio of the area of tympanic membrane perforation to the area of the tympanic membrane. Twelve standardized patients and 60 medical students were involved to assess the area of tympanic membrane perforation, in particular, the percentage of perforation size. Methods In total, 60 student doctors (including year 5 and year 6 medical students, intern and post-graduate year training of doctors) were recruited during their rotation at the Otolaryngology department of Taipei Medical University Shuang-Ho Hospital. Twelve standardized patients with chronic otitis media were recruited through a single otology practice. Oto-endoscopic examination was performed for all patients by using a commercially-available digital oto-endoscope, and clinical images of the tympanic membrane perforation were obtained. To demonstrate the variability of perforation size estimation by different student doctors, we calculated the percentage of perforation using the smartphone-based application for 12 tympanic membranes objectively and compared the results with those visually estimated by the 60 student doctors subjectively. Results The variance in the visual estimation by the 60 student doctors was large. By contrast, variances in smartphone-based application calculations were smaller, indicating consistency in the results obtained from different users. The smartphone-based application accurately estimated the presence of perforation for tympanic membranes with high consistency. The differences in visual estimations can be considerably great and the variances can be large among different individuals. Conclusions The smartphone-based application is a dependable tool for precisely evaluating the size of tympanic membrane perforation.