A biosensor scheme that employs long range surface plasmons (LRSPs) for the efficient excitation and collection of fluorescence light from fluorophore-labeled biomolecules captured in a three-dimensional hydrogel matrix is discussed. This new approach to plasmon-enhanced fluorescence (PEF) is experimentally and theoretically investigated by using the Kretschmann configuration of attenuated total reflection (ATR) method. A layer structure supporting LRSPs that consists of a low refractive index fluoropolymer layer, a thin gold film and a large binding capacity N-isopropylacrylamide (NIPAAm)-based hydrogel matrix swollen in an aqueous sample is employed. By using this layer architecture, the extended field of LRSPs probes the binding of biomolecules in the binding matrix at up to micrometer distances from the gold surface. With respect to regular surface plasmon-enhanced fluorescence spectroscopy (SPFS) and surface plasmon-coupled emission (SPCE), a narrower angular distribution of the fluorescence light intensity, a larger peak intensity and the excitation and emission at lower angles were observed.