To evaluate tympanic membrane regeneration therapy (TMRT) for pediatric tympanic membrane perforations (TMPs). Intervention study. Research institute hospital. In this study, 20 patients with chronic TMP (M/F: 13/7, 13/8 ears, age 0-15 years) treated with TMRT were evaluated. As comparison, 20 pediatric patients with chronic TMP who underwent myringoplasty/tympanoplasty were included. For the TM repair procedure, the edge of the TMP was disrupted mechanically, and gelatin sponge immersed in basic fibroblast growth factor was placed inside and outside the tympanic cavity and covered with fibrin glue. The TMP was examined 4 ± 1 weeks later. The protocol was repeated up to four times until closure was complete. Closure of the TMP and hearing improvement were evaluated at 16 weeks after the final regenerative procedure. Adverse events were monitored. The mean follow-up period was 427.1 days. The TM regenerated in all cases, but pinhole reperforation occurred in two cases, and the final closure rate was 90.5% (19 of 21). Hearing improved to 24.9 ± 7.6 dB on average before surgery and to 13.8 ± 5.4 dB after surgery. The AB gap improved from 12.9 ± 8.0 to 5.2 ± 3.5 dB.The myringoplasty/tympanoplasty group had significantly lower AB gap improvement compared with the TMRT group. There were no adverse events. TMRT can be expected to regenerate near-normal TMs with a high closure ratio, resulting in better-hearing improvement compared with the myringoplasty/tympanoplasty group, and is an effective treatment for children with long life expectancy.