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Purpose To compare the three-dimensional (3D) heads-up surgery with the traditional microscopic (TM) surgery for various vitreoretinal diseases. Methods A medical record review of patients that underwent 3D heads-up or TM vitreoretinal surgeries was performed from May 2020 to October 2021 in this retrospective case–control study. Main outcome measures included surgery-related characteristics, efficacy, safety, and satisfaction feedback from the surgical team. Results A total of 220 (47.6%) and 242 (52.4%) eyes were included in the 3D and TM groups, respectively. The 3D heads-up system significantly benefits delicate surgical steps, like the epiretinal membrane (ERM) peeling for ERM and internal limiting membrane peeling for idiopathic macular holes (P P = 0.049), while no difference by TM surgery (P = 0.45). For the satisfaction feedback, the 3D heads-up system was rated significantly higher in most subscales and the overall score (P P P > 0.05). Conclusion The efficacy and safety of the 3D heads-up surgery were generally comparable to the TM surgery. The 3D heads-up system could significantly benefit delicate surgical steps and achieve better surgical team satisfaction.