Purpose <p>To evaluate the efficacy and safety of subretinal injection of balanced salt solution (BSS) combined with vitrectomy and internal limiting membrane (ILM) peeling for managing large-diameter macular holes (MHs ≥ 400 μm).</p> Methods <p>A prospective interventional study was conducted on 31 eyes of 31 patients with full-thickness MHs (including 5 refractory MHs). Patients underwent 25 G vitrectomy, ILM peeling, followed by subretinal BSS injection using a 41 G needle. Outcomes were assessed at 1 and 3 months postoperatively with optical coherence tomography (OCT), best corrected visual acuity (BCVA), and microperimetry. Metrics of the microperimetry included macular integrity index (MII) and macular threshold (MT). Ten eyes completed 6 month followup. Changes in BCVA (LogMAR), MII, and MT before and after surgery, as well as MH closure and complications were compared and analyzed. Functional outcomes were analyzed only in eyes with successful type I closure.</p> Results <p>At 1 month, 93.54% (29/31) achieved complete MH closure. BCVA improved from 1.34 ± 0.59 preoperatively to 0.67 ± 0.20 at 1 month and 0.48 ± 0.36 at 3 months (<i>p</i> &lt; 0.001). MII improved from 98.77 ± 4.83 to 96.23 ± 11.38 (<i>p</i> = 0.015) and MT from 17.53 ± 5.04 to 22.54 ± 3.91 dB (<i>p</i> &lt; 0.001) at 3 months. In 10 eyes with 6 month followup, BCVA further improved to 0.41 ± 0.30 and MT to 24.64 ± 2.35 dB (both<i>p</i> &lt; 0.05). The refractory subgroup (<i>n</i> = 5) achieved 100% complete closure. No complications were observed during the follow-up time.</p> Conclusion <p>Combined 41 G subretinal BSS injection with PPV and ILM peeling is effective and safe for treating largediameter MHs (≥400 μm), achieving high anatomical closure rates and sustained visual functional improvement up to 6 months. Preliminary outcomes in refractory MHs are favorable; larger cohorts are needed for validation.</p>