Abstract: Objective To investigate the changes of microRNA-15 a(miR-15 a), microRNA-16(miR-16), microRNA-20 b(miR-20 b) and their relationship with diabetic retinopathy(DR) in patients with type 2 diabetes mellitus(T2 DM). Methods A total of 105 patients with T2 DM admitted to Lishui People's Hospital from May 2017 to May 2019 were divided into non-DR group(32 cases), non-proliferative diabetic retinopathy(NPDR) group(39 cases) and proliferative diabetic retinopathy(PDR) group(34 cases), and 40 cases of simple cataract patients in the same period were selected as control group. The levels of miR-15 a, miR-16 and miR-20 b in aqueous humor were detected by direct determination of miR-derived fragment length polymorphism(miRFLP). The levels of miR-15 a, miR-16 and miR-20 b in serum were measured by real-time fluorescence quantitative PCR(qRT-PCR). The predictive value of miR-15 a, miR-16 and miR-20 b levels in aqueous humor and serum for PDR was analyzed by ROC curve. Results The levels of miR-15 a, miR-16 and miR-20 b in aqueous humor and serum were PDR group, NPDR group, non-DR group and control group respectively, with significant differences(all P<0.05). There was a positive correlation between aqueous humor and serum levels of miR-15 a, miR-16 and miR-20 b in DR patients(all P<0.05). The area under the curve of predicting PDR was 0.912 and the sensitivity was 91.20%, the area under the curve of predicting PDR was 0.931 and the specificity was 92.30%, the predictive value was better than that of single application. Conclusion The increased levels of miR-15 a, miR-16 and miR-20 b in aqueous humor and serum of patients with T2 DM are associated with DR.