Chronic tympanic membrane (TM) perforation is a tubotympanic disease caused by either traumatic injury or inflammation. A recent study demonstrated significant progress in promoting the regeneration of chronic TM perforations through the application of nanofibers with radially aligned nanostructures and controlled release of growth factors. However, radially aligned nanostructures with stem cell-stimulating factors have never been used. In this study, insulin-like growth factor binding factor 2 (IGFBP2)-incorporated radially aligned nanofibrous patches (IRA-NFPs) were developed and applied to regenerate chronic TM perforations. The IRA-NFPs were prepared by electrospinning 8 wt% polycaprolactone in trifluoroethanol and acetic acid (9:1). Random nanofibers (RFs) and aligned nanofibers (AFs) were successfully fabricated using a flat plate and a custom-designed circular collector, respectively. The presence of IGFBP2 was confirmed via Fourier transform infrared spectroscopy, and the release of IGFBP2 was sustained for up to 20 days. In vitro studies revealed enhanced cellular proliferation and migration on AFs compared to RFs, with IGFBP2 incorporation further promoting these effects. Quantitative real-time PCR revealed mRNA downregulation, correlating with accelerated migration and increased cell confluency. In vivo studies using Choung’s COM model 1 showed IGFBP2-loaded RF and AF patches increased regeneration success rates by 1.59-fold and 2.23-fold, respectively, while also reducing healing time by 2.5-fold compared to controls. Furthermore, IGFBP2-incorporated AFs demonstrated superior efficacy in healing larger perforations with enhanced histological similarity to native TMs. This study, combining stem cell stimulating factors and aligned nanostructures, proposes a novel approach potentially replacing conventional surgical methods for chronic TM perforation regeneration.