PURPOSE: The purpose of this study was to analyze the macular vessel density layer-by-layer and compare the diagnostic value of each in diagnosing glaucoma. METHODS: This was a prospective comparative cross-sectional study, and the setting was glaucoma referral practice. The study participants were patients with primary open-angle glaucoma undergoing treatment with drugs, and age-matched normal controls who visited our clinic for regular eye examinations for refractive errors. All participants were investigated using macular optical coherence tomographic angiography, fundus photography, and 24-2 visual field (VF) testing. Average vessel densities in the retinal nerve fiber-ganglion cell-inner plexiform layer (IPL), retinal nerve fiber-ganglion cell layer (GCL), retinal nerve fiber layer (RNFL), ganglion cell-IPL, GCL, and IPL segments on optical coherence tomographic angiography. RESULTS: Fifty-eight glaucomatous eyes of 58 participants and 52 healthy eyes of 52 normal subjects were included in the study. The average vessel densities of all segments, except the RNFL in the glaucoma group, were significantly lower than that in normal subjects. The average vessel density in the ganglion cell-IPL showed the highest correlation with the mean deviation and VF index of the VF ( = 0.515 and 0.538, respectively) and the best area under receiver operating characteristic curve to discriminate between patients with glaucoma and patients with normal eyes (0.750). CONCLUSIONS: The present study demonstrated that macular vessel density in the ganglion cell-IPL has a higher diagnostic ability and better correlation with functional damage in glaucoma than that in the superficial vascular plexus. TRANSLATIONAL RELEVANCE: These findings suggest that the macular vessel density in the ganglion cell-inner plexiform layer is better than that in the conventional superficial vascular plexus for detecting glaucoma.