Proliferative diabetic retinopathy (PDR) is the most severe complication of chronic hyperglycaemi stimulates oxidative stress that changes the retinal basement membrane function and provokes neovascularization, macular edema and retinal detachment. But an oxidative–antioxidant biomarker assessment in ocular matrices, such as aqueous humor (AH) and vitreous, might show the oxidative stress (OS) status in the posterior segment. Here, we show a cross-sectional analytical study of 39 patients who had a vitrectomy and assess the levels of different oxidative–antioxidant biomarkers in blood, aqueous and vitreous humor in three groups: diabetes mellitus 2 (DM2) with PDR [DM(+)PDR(+)] (n =13), DM2 without PDR [DM(+)PDR(−)] (n = 13) and non-DM2 non-PDR [DM(−)PDR(−)] as the control group (n = 13). Our finding suggests the presence of oxidative stress in diabetic retinopathy, as evidenced by increased levels of 8-isoprostanes and decreased levels of total antioxidant capacity from stages before the development of diabetic retinopathy. Our results reveal a notable increment in catalase levels in the DM(+)PDR(+) group in blood and vitreous humor. Likewise, we identified that the DM(+)PDR(−) group presents significant levels in 8-IP and SOD in vitreous humor and blood versus aqueous humor. These finding suggest the role of antioxidant enzymes in compensating oxidative stress mechanisms in PDR development.