Objective: To assess the repeatability and reproducibility in measurements of retinal vessel diameter and tortuosity in diabetic patients using the Automated Retinal Image Analyzer (ARIA). Methods: A prospective diagnostic trial was performed; 2 skilled researchers performed a semi-automatic analysis of retinal vessels in digital color fundus photography (45° field of view centered on the optic disc) from 49 diabetic patients using ARIA software. Six largest arterioles and venules coursing through an area of 0.5–1.0 disc diameter surrounding the optic disc were selected. Then the retinal vessel parameters were calculated into the central retinal artery equivalent (CRAE), central retinal vein equivalent (CRVE), arteriole-to-venule ratio (AVR), mean retinal arteriolar tortuosity (MRAT) and mean retinal venular tortuosity (MRVT). To evaluate the repeatability of those values in 2 measurements by the same researcher and the reproducibility between different researchers. Bland-Altman analysis was employed to assess the agreement of the outcomes. Results: There was no significant difference in the CRAE, CRVE, and AVR values in the two-time measurements of researcher A (t= -0.089, -1.425 and 1.214, all P>0.05). It also showed a good repeatability and a highly positive correlation (ICC≥0.725, r≥0.721). There was no significant difference in those measurements between researcher A and B (t= -1.169, -0.050, 0.941, all P>0.05). It also showed a good reproducibility and a highly positive correlation (ICC≥0.750, r≥0.697). Results showed that there was no significant difference in the MRAT and MRVT values both in intraobserver and interobserver measurements (P≥0.05). Besides, Spearman’s nonparametric correlation analysis showed good repeatability and reproducibility (r≥0.571, r≥0.588, all P<0.01). Conclusion: ARIA software yields good repeatability and reproducibility in the measurements of retinal vessel diameter and tortuosity in diabetic patients.