Diabetic retinopathy (DR) is the most common cause of blindness in people under the age of 65. Unfortunately, the current screening process for DR restricts the population that can be evaluated and the disease goes undetected until irreversible damage occurs. Herein, we aimed to evaluate homocysteine (Hcy) as a biomarker for DR screening. Hcy levels were measured by enzyme-linked immuno sorbent assay (ELISA) and immunolocalization methods in the serum, vitreous and retina of diabetic patients as well as in serum and retina of different animal models of DM representing type 1 diabetes (streptozotocin (STZ) mice, Akita mice and STZ rats) and db/db mice which exhibit features of human type 2 diabetes. Our results revealed increased Hcy levels in the serum, vitreous and retina of diabetic patients and experimental animal models of diabetes. Moreover, optical coherence tomography (OCT) and fluorescein angiography (FA) were used to evaluate the retinal changes in mice eyes after Hcy-intravitreal injection into normal wild-type (WT) and diabetic (STZ) mice. Hcy induced changes in mice retina which were aggravated under diabetic conditions. In conclusion, our data reported Hcy as a strong candidate for use as a biomarker in DR screening. Targeting the clearance of Hcy could also be a future therapeutic target for DR.