We designed a microfluidic immunosensor based on the principle of insulator-based dielectrophoretic (iDEP) and electrowetting-on-dielectric (EWOD). We fabricated a 30-μm-thick SU-8 film containing microapertures placing between two EWOD-driven droplets to create a nonuniform applied electric field. With such a device, we can separate beads with different sizes by iDEP. Furthermore, the detection of protein, fluorescent-labeled streptavidin specimens, in diluted suspension by biotin-labeled polystyrene beads can also be carried out. We used EWOD technique to make biotin detectable protein, streptavidin, by means of affinity which forms a strongest non-covalent bond among them. Our device provides a simple, portable, effective means of particles focusing with diluted suspension, according to the uncomplicated fabrication process by photolithography and the large surface-to-volume ratio of biotin-labeled polystyrene beads on micro scale. With this technique, we can decrease the detection time and concentration limit of the specimens with a small volume of reagent consumed.