Purpose:To investigate how long indocyanine green (ICG) remains in the fundus after vitreoretinal surgery assisted with ICG, and to identify factors that influence the persistence duration. Methods: Fifty five eyes diagnosed as idiopathic macular hole (Stage 2 and 3) were randomly divided into five groups. ICG solution at concentrations of 5, 2.5, 2.5, 1.25, and 0.5 mg/ml, employed in cases of Group Ⅰ to Ⅴ respectively, was applied to stain the internal limiting membrane (ILM) during the procedure of internal limiting membrane peeling. A prospective study was carried out after pars plana vitrectomy and ILM peeling were performed on 55 eyes with Stage 2, 3, or 4 idiopathic macular holes. Infrared fundus pictures were obtained in all patients before and after surgery. Results: High levels of fluorescence from residual ICG (ICG hyperfluorescence) were mainly localized at the posterior pole of the fundus after surgery. In Group Ⅰ,Ⅱ,Ⅲ,Ⅳ and Ⅴ, the duration of persistence of flurorescence from ICG was 8.33±0.87, 3.59±0.94, 3.75±0.79, 2.30±0.48, and 1.29± 0.49 months, respectively. Although no significant difference was detected between Group Ⅱ and Group Ⅲ, the general inter-group difference was significant among the five groups in which different ICG concentration was applied. In Group Ⅲ, even though 90% of the macular holes acquired anatomical closure, ICG hyperfluorescence was detected in the macular area. Conclusion:ICG remains in the fundus for a period of months. The persistence duration of fluorescence from ICG is positively correlated with the concentration and the staining time of ICG. Hyaluronan is beneficial in reducing the amount of ICG residue in the macular area.