By integrating photopolymerized cross-linked polyacrylamide gels within a microfluidic device, we have developed a microanalytical platform for performing electrophoresis-based immunoassays. The microfluidic immunoassays are performed by gel electrophoretic separation and quantitation of bound and unbound antibody or antigen. To retain biological activity of proteins and maintain intact immune complexes, nondenaturing polyacrylamide gel electrophoresis conditions were investigated. Both direct (noncompetitive) and competitive immunoassay formats are demonstrated in microchips. A direct immunoassay was developed for detection of tetanus antibodies in buffer as well as diluted serum samples. After an off-chip incubation step, the immunoassay was completed in less than 3 min and the sigmoidal dose-response curve spanned an antibody concentration range from 0.17 to 260 nM. The minimum detectable antibody concentration was 0.68 nM. A competitive immunoassay was also developed for tetanus toxin C-fragment by allowing unlabeled and fluorescently labeled tetanus toxin C-fragment compete to bind to a limited fixed concentration of tetanus antibody. The immunoassay technique described in this work shows promise as a component of an integrated microfluidic device amenable to automation and relevant to development of clinical diagnostic devices.