Bead-based immunosensing is a promising technology in the point-of-care diagnosis due to its high flexibility. To realize rapid and selective diagnosis, an optoelectrokinetic platyeform, termed rapid electrokinetic patterning (REP), was employed herein to drive a bead-based fluorescent immunosensor for diagnosis of diabetic retinopathy by lipocalin 1 (LCN1) and vascular epithelial growth factor (VEGF). The measurement was made in an aqueous droplet sandwiched between two parallel electrodes. With an electric field and a focused laser beam applying on the microchip simultaneously, the immunocomplexes in the droplet were further concentrated to enhance the fluorescent signal. The limit of detection (LOD) reached nearly 110 pg/mL. A DR phase plot was made to provide a clear way to determine whether a tested subject was under the threat of DR. At last, a preclinical evaluation was conducted. The accuracy of the proposed technique reached 94.74% in PDR and 89.47% in DM. The optoelectrokinetically driven bead-based immunosensor enables a noninvasive measure to the early diagnosis of diabetic retinopathy.