In this study, we developed a novel immunoassay method based on the magnetic force in microfluidic channel or lab-on-a-chip. The magnetic force-based microfluidic immunoassay was firstly devised and successfully applied to detect the rabbit IgG and mouse IgG as model analytes of sandwich immunoassay. In this experiment, we performed sandwich immunoassay using the fluorescent polystyrene microbeads, superparamagnetic nanoparticle solution as a label, and rabbit and mouse IgG as analytes. The microchannels of a microfluidic device were fabricated by polydimethylsiloxane (PDMS) molding processes and bonded on a slide glass by plasma treatment. The movements of microbeads conjugated with magnetic nanoparticles were demonstrated by magnetic field gradients in microfluidic channel and compared with calculated magnetic field gradients. Dual analyte detection of rabbit IgG and mouse IgG in a single reaction was carried out in a microfluidic device. The detectable range of concentration of rabbit IgG and mouse IgG was 244 pg/mL to 4 μg/mL and 62.5 ng/mL to 4 μg/mL, respectively. The lowest concentration of rabbit IgG and mouse IgG that was measured over the background was almost 244 pg/mL and 15.6 ng/mL, respectively. The multiplexed immunoassay using encoded microbeads which are immobilized with a certain antibody, respectively, could be possible using this detection principle. Moreover, with micro electromagnetic integrated systems using this detection principle, high sensitive, and low detection limited assay system could be developed.