The use of thermoresponsive poly(N-isopropylacrylamide)-based hydrogel (pNIPAAm) for rapid tuning of surface plasmon resonance (SPR) is reported. This approach is implemented by using an SPR layer architecture with an embedded indium tin oxide microheater and pNIPAAm film on its top. It takes advantage of rapid thermally induced swelling and collapse of pNIPAAm that is accompanied by large refractive index changes and leads to high thermo-optical coefficient of dn/dT = 2 × 10–2 RIU/K. We show that this material is excellently suited for efficient control of refractive index-sensitive SPR and that it can serve simultaneously as a 3D binding matrix in biosensor applications (if modified with biomolecular recognition elements for a specific capture of target analyte). We demonstrate that this approach enables modulating of the output signal in surface plasmon-enhanced fluorescence spectroscopy biosensors and holds potential for simple time-multiplexing of sensing channels for parallelized readout of fluorescence assays.