This research describes a high sensitivity microfluidic bead-based immunosensor based on the principle of insulator-based dielectrophoresis (iDEP). An insulator film with small holes between two electrodes creates a nonuniform electric field. By applying appropriate voltage and frequency, the fluorescent beads are concentrated to lower electric field regions due to the difference of dielectric properties. This concentrating step enhances the fluorescence intensity of analytes and decreases the detection limit of immunosenser. In this research, the fluorescence dye is conjugated with streptavidin which has high affinity to biotin. We use biotin-labeled polystyrene beads to bind with streptavidin, therefore, we can further detect fluorescent streptavidin conjugates by a fluorescence microscope. The biotin-labeled polystyrene beads perform not only various chemical characteristics by labeling different functional groups but also offer an increased surface area for antibodies or antigens to immobilize on. Finally, we fabricate a microfluidic bead-based immunosensor with high sensitivity (1 pg/ml), short analysis time (∼10 minutes), few sample consumption (∼0.5 μl) and without physical microchannel.