Long range surface plasmons (LRSPs) are optical waves that propagate along thin metallic films with up to orders of magnitude lower damping compared to regular surface plasmons. Therefore, LRSPs attracted a great deal of attention for development of ultrasensitive biosensors based on surface plasmon resonance and surface plasmon-enhanced fluorescence spectroscopy (SPFS). In this article, the authors investigate the excitation of LRSPs on a biosensor-compatible layer structure consisting of a Cytop fluoropolymer, thin gold film modified by thiol self-assembled monolayer for coupling of receptor biomolecules, and an aqueous sample on its top. The morphology, and optical and electrical properties of the layer structure are determined and related to the performance of a SPFS biosensor. Through increasing the surface energy of Cytop fluoropolymer by O-2 plasma, more compact gold films that exhibit lower roughness were prepared which resulted in a higher binding capacity, decreased nonspecific adsorption of biomolecules to the biosensor surface, and in a larger enhancement of electromagnetic field intensity accompanied with the excitation of LRSPs. The authors show that by improving the quality of a gold film supporting LRSPs, the fluorescence signal can be enhanced up to sixfold with respect to regular SPFS biosensors.