In this work, we report on the uniform deposition of tailored plasmonic coatings on polymer substrates and on the distance dependence of the plasmonic enhancement of a fluorescent dye. Silver, gold, and silver/gold alloy nanoparticles (NPs) with a range of diameters were synthesized using chemical techniques and characterized using UV-vis absorption spectroscopy, transmission electron microscopy (TEM), and atomic force microscopy (AFM). Reproducible polyelectrolyte (PEL) layers, which were deposited on plastic microwell plates using a layer-by-layer technique, served as both a stable and uniform substrate for deposition of the NPs as well as providing spacer layers of known thickness between the NPs and the fluorescent dye. A maximum enhancement factor of approximately 11 was measured for 60 nm diameter pure silver NPs, for a dye-NP separation of approximately 3 nm. A shift in the localized surface plasmon resonance (LSPR) wavelength as a function of the effective refractive index of the PEL layers was also observed, and the measured shifts show a similar trend with theoretical predictions. This work will contribute toward the rational design of optical biochip platforms based on plasmon-enhanced fluorescence.