AIM: To detect the concentrations of reactive oxygen species (ROS), transient receptor potential mucin-1 (TRPML1), and autophagy-related (Atg) proteins (LC3-I, LC3-II, and Beclin1) in vitreous humor of patients with simple rhegmatogenous retinal detachment (RRD). METHODS: RRD patients enrolled as the RRD group, and patients with idiopathic macular hole (IMH) and idiopathic macular epiretinal membrane (IMEM) were enrolled as control group. The levels of ROS, TRPML1, LC3-I, LC3-II, and Beclin1 in vitreous humor of patients in the RRD and control groups were detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: The RRD group included 28 eyes 28 patients and had a higher concentration of ROS in vitreous humor (631.86±18.05 vs 436.34±108.22 IU/mL, P<0.05). The ROS level in patients with a wide retinal detachment (RD) extent (RD range ≥1/2) was higher than that with a narrow RD extent (RD range<1/2, P<0.05). ROS concentration was negatively correlated with RD time (r=-0.46, P=0.01). The expression levels of LC3-I and Beclin1 significantly decreased in RRD (P<0.05), but there were no correlations with the RD time, RD extent, or macular involvement. CONCLUSION: In eyes with RRD, the concentration of ROS in vitreous humor increases and the expression levels of Atg proteins decrease, reflecting possibly that autophagy is inhibited.