Background To evaluate the effect of internal limiting membrane (ILM) peeling surrounding macular holes (MH) for the function of retina by microperimetry-3(MP-3). Methods This is a prospective, cohort study which included patients with MHs who were treated by 23-gauge 3-port pars plana vitrectomy and ILM peeling with air tamponade. Color fundus photography, retinal optical coherence tomography and MP-3 were performed 1 week before, 1 and 4 months after the operation. In MP-3 examination, a customized follow-up pattern with 45 spots in the central 8 degrees visual field was used. The spots corresponding to the retina surrounding macular holes were selected for comparison of pre- and post-operative function. Results We incuded 44 eyes of 44 patients with best corrected visual acuity (BCVA) of 1.06 +/- 0.40 (logMAR). All eyes achieved an anatomical success at 4 months. BCVA significantly improved at 1 month (0.53 +/- 0.30, P < 0.01) and 4 months (0.31 +/- 0.24, P < 0.01), respectively. Mean retinal sensitivity (MRS, dB) of the retina surrounding macular hole was 23.46 +/- 3.01 dB at baseline, and significantly increased at 1 month (26.25 +/- 2.31 dB, u = - 4.88, P < 0.01) and 4 months (27.14 +/- 2.45 dB, t = - 6.29, P < 0.01). Patients with increased MRS are significantly younger than those with deceased MRS (59.72 +/- 3.22 years vs. 65.60 +/- 8.19 years, P < 0.01). After ILM peeling, the increasing extent of MRS was significantly higher in inferior and nasal retina than in superior and temporal retina at 1 and 4 months (P < 0.05). Conclusion ILM peeling in normal retina will not decrease the retinal function in a short-term after surgery.