PURPOSE To investigate the efficacy of autologous serum irrigation for removal of retinal indocyanine green (ICG) dye used to visualize the internal limiting membrane (ILM) in macular hole surgery. METHODS Eighteen consecutive eyes of 16 patients with macular holes underwent vitrectomy and ILM peeling using ICG solution. Nine of 18 eyes underwent intravitreal autologous serum irrigation before completion of the surgery to remove residual ICG dye in the retina. The main outcome measures were postoperative visual acuity, macular hole status, retinal pigment epithelial changes, and time course of ICG dye fading from the retina with or without intravitreal autologous serum irrigation. RESULTS No significant difference was noted in the improvement of visual acuity between the two groups. An abnormal pigment epithelial change was seen in one eye without serum application. ICG dye disappeared from the retina within an average of 6.1 months after surgery without serum and 2.4 months after surgery with serum (P < 0.01). CONCLUSIONS Autologous serum irrigated in the vitreous cavity just before completion of macular hole surgery can help remove ICG dye used in surgery. The application was simple and safe and significantly shortened the period of residual retinal ICG staining.