This study reports an integrated microfluidic system for rapid immunosensing of antibodies associated with dengue fever by utilizing enzyme-linked immunoabsorbent assay (ELISA) poly(dimethylsiloxane) (PDMS)-based chip. In this study, we successfully developed a rapid immunosensing chip with pre-functionalized surfaces to avoid protein adsorption. The functionalized PDMS surface was found to be highly effective in reducing the adsorption time in microchannels. The functionalized surfaces can be stable at least for 1 month when stored at 4°C. In addition, a suction-type microfluidic transportation module was used to form a single-fluid-slug transportation and to mix the reagents for rapid detection of dengue virus. The entire process can be finished within 30 min. The sensitivity was found to be superior to the conventional methods.