This systematic review and meta-analysis evaluated the efficacy and safety of autologous human serum as an adjuvant agent in pars plana vitrectomy (PPV) for macular holes (MH). Thus, a comprehensive search was conducted across PubMed, Web of Science, Embase, and the Cochrane Library databases up to August 20th, 2023. The inclusion criteria targeted randomized clinical trials (RCTs) or non-RCTs that compared the use of autologous serum in vitrectomy for MH with the same procedure without the serum. The outcomes were MH closure rates and postoperative complications such as retinal detachment and cataracts. Odds ratios (OR) and mean differences (MDs) were calculated using a random-effects model. Review Manager 5.3 (The Cochrane Collaboration, Oxford, UK) was used for statistical analysis. Four studies, comprising two RCTs and two non-randomized cohort studies with 373 eyes of 372 patients, were included. The pooled analysis showed no significant difference in MH closure rates (OR 1.28; 95% confidence interval (CI): 0.48 to 3.43; P=0.62) and no difference concerning the incidence of adverse events (OR 0.97; 95% CI: 0.30-3.09; P=0.96). Leave-one-out sensitivity analysis excluding the study by Lauritzen et al. revealed a significant difference in anatomical closure, favoring the serum arm, and demonstrated a reduction in the level of heterogeneity. Our meta-analysis demonstrated no difference between groups in the pooled analysis of all studies. However, considering the quality assessment of one of the included studies, and observing the divergent result in sensitivity analysis following its exclusion, there are indications that might suggest the superiority of the serum in terms of the analyzed endpoints. This finding highlights the existing research gaps and the imperative need for additional high-quality randomized trials to further investigate this treatment.