Negative dielectrophoresis (n-DEP) have been used to manipulate microparticles with immunoreagents (antigens or antibodies) in a microfluidic channel, and applied to develop a rapid immunoassay system. A microfluidic device, with three-dimensional (3-D) microelectrodes fabricated on two substrates, was used to manipulate particle flow in the channel and to capture the particles in the caged area that was enclosed by the collector electrodes. Polystyrene microparticles (6 μm diameters) modified with anti-mouse immunoglobulin G (IgG) were manipulated and captured in the caged area by using n-DEP. A sandwich immunoassay was achieved by successively injecting a sample solution containing mouse antigen (IgG), and a solution containing FITC-labeled anti-mouse IgG antibody, into the channel. The fluorescence intensity from captured particles in the caged area increased with increasing concentrations (10 ng/ml to 10 μg/ml) of mouse IgG. The described system enables mouse IgG to be assayed in 40 min. This immunosensing system using the n-DEP technique is faster and simpler than conventional enzyme-linked immunosorbent assay (ELISA) using microtiter plates, and has the significant advantage that sensing requires simple and easy handling since unreacted immunomolecules are flushed from the signal detection area by the fluidic stream. The device can be reused by removing the microparticles. The automatic separation of free fractions from desired analytes and labeled antibodies can be achieved using a microfluidic device based on n-DEP.