We have realized fluorescence polarization immunoassay (FPIA) on a microchip in about 1 minute. FPIA is a homogeneous competitive immunoassay which is based on measuring fluorescence polarization after competitive binding of an analyte and a tracer to an antibody. We constructed a microfluidic FPIA system composed of a newly designed microchip, a laser, a CCD camera and an optical microscope with two specially installed polarizers-one fixed and one rotatable. Theophylline, a typical small drug molecule, was used as a model analyte. Theophylline and fluorescence-labeled theophylline were introduced through different inlets and combined in a 100 microm-wide microchannel where anti-theophylline antibody was added. To optimize the microchip design for FPIA, we investigated the diffusion time of theophylline and the mixing time of theophylline and antibody in this channel, which were 6 s and 36 s, respectively. We successfully carried out a quantitative analysis of theophylline in serum near the therapeutic range in 65 s. In FPIA, a larger tracer-antibody complex emits more polarized fluorescence than the tracer, and therefore, by increasing the antigen concentration in a sample, more polarization relaxation is observed since the tracer-antibody complex concentration is decreased and the tracer concentration is increased. Tracer binding to an antibody is directly measured by spectroscopic techniques without any separation process.This microchip-based FPIA is very simple and rapid, unlike microchip-based heterogeneous immunoassay, because it does not require several processes such as washing and reflowing and immobilizing of antibodies or antigens in the channel. In the future, microchip-based FPIA should find frequent use for point-of-care testing in the clinical field, where conventional FPIA has been used for laboratory tests.