Object To establish a method for visualizing and quantifying the abnormal expression of vascular endothelial growth factor (VEGF) in the retina ，and to help the early detection and treatment of diabetic retinopathy (DR). Methods Immunofluorescence staining and imaging were performed on the rat retinal flatmounts. VEGF and microvessels were segmented, extracted and reconstructed. Quantitative parameters such as positive VEGF units, microvascular density and fractal dimension were used and the differences between the 8-week diabetic retinopathy model group (n=10) and the normal control group (n=10) were compared. Results In the DR model, there was colocalization between the high expression region of VEGF probe and the abnormal region of vascular, and VEGF accumulates on the surface of retinal microvascular bifurcation or swollen vessel wall. The fractal dimension, density and curvature of microvessels in DR group were significantly higher than those in normal control group (P<0.05). The PU value of VEGF in DR group was significantly higher than that in comparison group (P<0.05). There was no significant difference in microvascular void (P>0.05). Conclusions In this study, a quantitative evaluation method of VEGF is proposed, which can reflect the abnormal distribution of VEGF in the retina of DR model qualitatively and quantitatively, and provide a quantitative and visual way for the research of anti-VEGF drugs.