Bead-based immunosensing has been growing as a promising technology in the point-of-care diagnostics because of great flexibility. For dilute samples, functionalized particles can be used to collect dispersed analytes and act as carriers for particle manipulation. To carry out rapid and selective diagnosis, a bead-based optoelectrokinetic immunosensing technique was developed herein to detect biomarkers, lipocalin 1 (LCN1) and TNF-α, for diabetic retinopathy (DR). The measurement was made in a sample droplet sandwiched between two parallel electrodes. With an electric field and a focused laser beam simultaneously applying on the microchip, the immunocomplexes in the droplet were further concentrated within the region of irradiation to enhance the fluorescent signal. The optoelectrokinetic platform, termed rapid electrokinetic patterning (REP), is excellent in dynamic and programmable particle manipulation. Therefore, the detection could be complete in roughly 10 s. With an appropriate frequency modulation, the two DR biomarkers were detected at a time. The limit of detection (LOD) of the REP-enabled measurement reached as low as 100 pg/mL. The combined use of bead-based immunoassays and the optoelectrokinetic platform therefore provides an insightful measure to the early diagnosis of diseases.