We assess the level of tumour necrosis factor alpha (TNF-alpha) in tear fluids and other serum parameters associated with diabetes in different degrees of diabetic retinopathy. We have performed a prospective, nonrandomized, observational study. Study population consisted of 16 healthy subjects (controls) and 32 type 2 diabetic patients: 16 affected by proliferative diabetic retinopathy (PDR) and 16 with nonproliferative retinopathy (NDPR, background/preproliferative). Body mass index, urinary albumin, blood glucose, HbA1c, and tear levels of TNF-alpha were measured in all subjects. The value of glycaemia, microalbuminurea, and Body mass index in diabetic retinopathy groups were higher than those in control group (P < 0.05). Glycemia in NPDR: 6.6 mmol/L (range: 5.8–6.3); in PDR: 6.7 mmol/L (range: 6.1–7.2); in control: 5.7 mmol/L (range: 4.9–6.1); microalbuminurea in NPDR: 10.6 mg/L (range: 5.6–20); in PDR: 25.2 mg/L (range: 17–40); in control: 5.3 mg/L (range: 2.6–10); Body mass index in NPDR: 26 Kg/m2 (range: 20.3–40); in PDR: 28 Kg/m2 (range 20.3–52); in control: 21 Kg/m2 (range 19–26). The TNF-alpha concentrations in tears increase with the severity of pathology and were lower in control group than in diabetic subjects. In the end, the level of TNF-alpha is highly correlated with severity of diabetic retinopathy and with nephropathy. Tear fluid collection may be a useful noninvasive method for the detection of proliferative diabetic retinopathy.