Red blood cell (RBC) deformability is a critical biophysical property that enables effective passage of RBCs through microvasculature and ensures proper oxygen delivery. Impairment of this property is associated with various pathological conditions, including type 2 diabetes mellitus (T2DM). In this study, we developed an automated microfluidic platform for high-throughput and real-time assessment of RBC deformability under controlled flow conditions. The device features a structured microchannel design and integrated imaging to quantify individual cell deformation responses. Comparative analyses of RBCs from healthy individuals and T2DM patients revealed significant reductions in deformability in the diabetic group. In vivo validation using a diabetic mouse model further confirmed the progressive decline in RBC deformability under chronic hyperglycemia. This microfluidic approach provides a robust and efficient tool for characterizing RBC mechanical properties, offering potential for disease monitoring and clinical diagnostic applications.