ZNF503 antisense RNA 1 (ZNF503-AS1) is a newly identified long non-coding RNA (lncRNA) that regulates retinal pigment epithelium differentiation. To study its role in diabetic retinopathy, we performed RT-qPCR to measure plasma ZNF503-AS1 levels of 298 diabetic patients immediately after the diagnosis, during the follow-up, and at the end of follow-up. Plasma lncRNA ZNF503-AS1 expression in 96 healthy participants was also detected by RT-qPCR. Transforming growth factor beta 1 (TGF-β1) expression after ZNF503-AS1 overexpression was detected by Western blot. Cell proliferation and apoptosis were detected by cell proliferation and apoptosis assays, respectively. We found that ZNF503-AS1 was not differentially expressed in healthy participants and diabetic patients. High plasma lncRNA ZNF503-AS1 level was correlated with a high incidence of diabetic retinopathy. Plasma lncRNA ZNF503-AS1 level was higher in patients with diabetic retinopathy than in patients with other complications (p < 0.05). ZNF503-AS1 overexpression inhibited proliferation, promoted cell apoptosis, and upregulated TGF-β1 expression (p < 0.05). We concluded that ZNF503-AS1 might participate in diabetic retinopathy by activating TGF-β signaling.