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Abstract Background The goal was to characterize retinal vasculature by quantitative analysis of arteriole-to-venule (A/V) ratio and vessel density in fundus photos taken with the PanOptic iExaminer System. Methods The PanOptic ophthalmoscope equipped with a smartphone was used to acquire fundus photos centered on the optic nerve head. Two fundus photos of a total of 19 eyes from 10 subjects were imaged. Retinal vessels were analyzed to obtain the A/V ratio. In addition, the vessel tree was extracted using deep learning U-NET, and vessel density was processed by the percentage of pixels within vessels over the entire image. Results All images were successfully processed for the A/V ratio and vessel density. There was no significant difference of averaged A/V ratio between the first (0.77 ± 0.09) and second (0.77 ± 0.10) measurements (P = 0.53). There was no significant difference of averaged vessel density (%) between the first (6.11 ± 1.39) and second (6.12 ± 1.40) measurements (P = 0.85). Conclusions Quantitative analysis of the retinal vasculature was feasible in fundus photos taken using the PanOptic ophthalmoscope. The device appears to provide sufficient image quality for analyzing A/V ratio and vessel density with the benefit of portability, easy data transferring, and low cost of the device, which could be used for pre-clinical screening of systemic, cerebral and ocular diseases.