<p>We present a rapid and highly sensitive immunoassay platform based on an ultra-thin immuno-wall microfluidic device with an easy-to-perform sequential fluorescence signal increment method. The ultra-thin immuno-wall was fabricated using a special type of water-soluble photopolymer mixed with streptavidin via photolithography. During photolithography, the photopolymer formed a three-dimensional cross-linked structure, and streptavidin was immobilized in the cross-linked structure based on the click chemistry reaction. The immobilized streptavidin was used to immobilize biotin-conjugated antibodies on the cross-linked structure to capture biomarkers, forming immune complexes on the surface, known as an “immuno-wall.” A sequential fluorescence signal increment method utilizes two different fluorescence-labeled antibodies with high affinity that were incubated several cycles in the immuno-wall to enhance the fluorescence signal. Moreover, an ultra-thin immuno-wall was developed to reduce the nonspecific binding and increase the signal-to-noise ratio. To evaluate the performance of this immunoassay platform, the spike protein from the SARS-CoV-2 virus was selected as the target biomarker. This immunoassay platform exhibited a limit of detection of 0.01 ng/mL, and the detection time was 30 min, which is comparable to rapid antigen tests. This immunoassay platform demonstrates significant potential for early-phase disease diagnosis.</p>