We report an enzyme-link immunosorbent assay (ELISA) based on patterned pseudopaper that is made of photonic nitrocellulose for highly sensitive fluorescence bioanalysis. The pseudopaper is fabricated using self-assembled monodisperse SiO2 nanoparticles that are patterned on a polypropylene substrate as template. The self-assembled nanoparticles have a close-packed hexagonal (opal) structure, so the resulting nitrocellulose has a complementary (inverse opal) photonic structure. Owing to the slow-photon effect of the photonic structure, fluorescence emission for ELISA is enhanced by up to 57-fold without increasing the assay time or complexity. As the detection signal is significantly amplified, a simple smartphone camera suffices to serve as the detector for rapid and on-site analysis. As a demonstration, human IgG is quantitatively analyzed with a detection limit of 3.8 fg/mL, which is lower than that of conventional ELISA and paper-based ELISA. The consumption of sample and reagent is also reduced by 33 times compared with conventional ELISA. Therefore, the pseudopaper ELISA based on patterned photonic nitrocellulose is promising for sensitive, high-throughput bioanalysis.