We report a novel biosensor platform based on surface plasmon-enhanced fluorescence spectroscopy (SPFS) and a responsive N-isopropylacrylamide (NIPAAm) hydrogel binding matrix. This binding matrix highly swells in aqueous environment and it can be modified with receptor biomolecules by using active ester coupling chemistry. After the binding of target analyte molecules contained in a sample by receptor biomolecules immobilized in the hydrogel matrix, the captured analyte molecules can be compacted on the surface through the collapse of the gel triggered by an external stimulus. A thin hydrogel NIPAAm-based film was attached to a gold sensor surface and modified with mouse IgG receptor molecules. The affinity binding of antibodies against mouse IgG that were labeled with Alexa Fluor chromophores was observed by surface plasmon-enhanced fluorescence spectroscopy. We demonstrate that the collapse of the hydrogel matrix results in the enhancement of measured fluorescence intensity owing to the increase in the concentration of captured molecules within the evanescent field of surface plasmons.