Diabetic retinopathy (DR) is an ocular morbidity that usually occurs in the retina of patients with chronic diabetes. Without proper medical intervention, DR eventually may lead to blindness. To meet the clinical need, this study developed an immunosensing technique based on optoelectrokinetically enabled beads for simultaneously detecting dual biomarkers, lipocalin 1 (LCN1) and tumor necrosis factor-α (TNF-α). A sandwiched configuration comprising capture antibody–antigen-probe antibody was formed by conjugating particles with capture antibodies. The additional dye-labeled secondary antibodies were eventually attached to the probe antibodies to determine the presence of the target biomarkers. The concentration of the target biomarkers was measured based on fluorescence intensity. The optoelectrokinetic technique, named as rapid electrokinetic patterning (REP), was used to enhance the fluorescence signal by concentrating the functionalized particles into a cluster. REP exhibited limit of detection as low as 110 pg/mL and was operated with simultaneous applications of optical illumination and electric field. Particles of different sizes were labeled with different dye colors to detect the corresponding DR biomarkers. The efficiency and accuracy of the proposed technique were increased by cross-matching multiple biomarkers in the tear of patients with DR. The optoelectrokinetic system can be used for treatments performed by ophthalmologists and can be extended to diagnose other oculopathies in the future.