The aim of this prospective study was to evaluate the changes in stereopsis in patients who underwent vitrectomy for macular hole (MH) and assess the relationship between stereopsis and retinal microstructures. Fifty-two patients who underwent successful vitrectomy for unilateral MH and 20 control participants were recruited. We examined stereopsis using the Titmus Stereo Test (TST) and TNO stereotest (TNO), optical coherence tomography, and best-corrected visual acuity measurements, preoperatively, and at 3, 6, and 12 months postoperatively. As a result, preoperative and postoperative 3, 6, and 12-month values of stereopsis assessed by TST (log) were 2.7, 2.2, 2.2, and 2.2, respectively. TNO (log) were 2.8, 2.5, 2.4, and 2.4, respectively. Stereopsis in MH after surgery was significantly worse than that in normal participants (p < 0.001). Preoperative TST significantly correlated with MH size and defect length of external limiting membrane (ELM). Postoperative TST demonstrated significant correlation with the preoperative ELM defect length, and postoperative TNO was associated with the preoperative interdigitation zone defect length. Vitrectomy for MH significantly improved stereopsis, although not to normal levels. The ELM defect lengths, which approximately correspond to TST circles, are prognostic factors for postoperative stereopsis by TST. The interdigitation zone defect length, similar in size to the TNO index, is a prognostic factor for postoperative stereopsis by TNO.