Ocular ischemic syndrome (OIS), attributable to chronic hypoperfusion caused by marked carotid stenosis, is one of the important factors that cause ocular neurodegenerative diseases such as optic atrophy. The current study aimed to detect blood flow perfusion in a visual pathway by arterial spin labeling (ASL) and magnetic resonance imaging (MRI) for the differential diagnosis of OIS. This diagnostic, cross-sectional study at a single institution was performed to detect blood flow perfusion in a visual pathway based on 3D pseudocontinuous ASL (3D-pCASL) using 3.0T MRI. A total of 91 participants (91 eyes) consisting of 30 eyes with OIS and 61 eyes with noncarotid artery stenosis-related retinal vascular diseases (39 eyes with diabetic retinopathy and 22 eyes with high myopic retinopathy) were consecutively included. Blood flow perfusion values in visual pathways derived from regions of interest in ASL images, including the retinal-choroidal complex, the intraorbital segments of the optic nerve, the tractus optics, and the visual center, were obtained and compared with arm-retinal circulation time and retinal circulation time derived from fundus fluorescein angiography (FFA). Receiver operating characteristic (ROC) curve analyses and the intraclass correlation coefficient (ICC) were performed to evaluate the accuracy and consistency. Patients with OIS had the lowest blood flow perfusion values in the visual pathway (all p < 0.05). The relative intraorbital segments of optic nerve blood flow values at post-labeling delays (PLDs) of 1.5 s (area under the curve, AUC = 0.832) and the relative retinal-choroidal complex blood flow values at PLDs of 2.5 s (AUC = 0.805) were effective for the differential diagnosis of OIS. The ICC of the blood flow values derived from the retinal-choroidal complex and the intraorbital segments of the optic nerve between the two observers showed satisfactory concordance (all ICC > 0.932, p < 0.001). The adverse reaction rates of ASL and FFA were 2.20 and 3.30%, respectively. 3D-pCASL showed that the participants with OIS had lower blood flow perfusion values in the visual pathway, which presented satisfactory accuracy, reproducibility, and safety. It is a noninvasive and comprehensive differential diagnostic tool to assess blood flow perfusion in a visual pathway for the differential diagnosis of OIS.