Objective: To measure the thickness of retinal ganglion cell layer and inner limiting membrane-retinal pigment epithelium layer in the macular region, and analyze its correlation with new-onset ischemic stroke. Methods: By using a prospective study, 30 healthy subjects (60 eyes) who underwent physical examination in Affiliated Hospital of Weifang Medical University from December 2021 to March 2022 were selected as the control group (Group A). Thirty age-matched patients with new-onset ischemic stroke admitted at the same time (60 eyes) were selected as the stroke group (Group B). The ganglioncelllayer (GCL) and innerlimitingmembrane-Retinalpigmentepithelium (ILM-RPE) were compared between the two groups. It included the thickness and mean GCL thickness/Mean Volume thickness of ILM-RPE (GCL/ILM-RPE thickness ratio). Results: A total of 120 eyes were included, with 60 eyes in group A and B. The average GCL thickness in group A was (83.5±4.7) μm, and the average GCL thickness in group B was (78.7±10.5) μm,and the difference between groups was statistically significant (P<0.001). The minimum thickness of GCL in group A was (80.4±4.4) μm, and the minimum thickness of GCL in group B was (70.7±16.3) μm, showing statistical significance(P<0.001). The thickness of the central subregion of ILM-RPE in group A was (247.5±19.8) μm, and the thickness of the central subregion of ILM-RPE in group B was (241.6±36.3) μm, and the difference was not statistically significant between two groups (P=0.393). The mean volumetric thickness of ILM-RPE in group A was (278.4±9.8) μm, and the mean volumetric thickness of ILM-RPE in group B was (278.5±15.3) μm, and the difference was not statistically significant (P=0.448). The GCL/ILM-RPE thickness ratio of group A was (0.300 3±0.012 2), and that of group B was (0.286 2±0.028 6), showing a statistical difference between groups (P<0.001). Conclusion: Ischemic stroke affects the GCL thickness and the thickness ratio of GCL/ILM-RPE, which may be related to new-onset ischemic stroke.