The plasma polymerization technique, which enables deposition of thin and pin-hole free films, was employed to fabricate multilayered substrates for fluorescence-based sensitive immunoassay. A glass slide was modified with a 200-nm-thick Ag layer as a metal mirror and further coated with plasma-polymerized films (PPFs) up to 120 nm thick as the optical interference layers. Cy3-labeled antibody was spotted on the resulting multilayered substrate and the fluorescence image was observed. The Cy3 fluorescence was highly enhanced on the multilayered substrate compared with that observed on the area without PPFs. The maximum fluorescence intensity was obtained when PPF thickness was 63 nm, showing a 30-fold enhancement of fluorescence compared with that obtained from the unmodified substrate. The multilayered substrates with PPF on which multiple proteins were immobilized were further utilized to perform immunoassays, resulting in specific and highly enhanced fluorescence detection.