In the past several years we have demonstrated the metal-enhanced fluorescence (MEF) and the significant changes in the photophysical properties of fluorophores in the presence of metallic nanostructures and nanoparticles. MEF is largely dependent on several factors, such as chemical nature, size, shape of the nanostructure, and its distance from the interrogating fluorophore. Herein, we elucidate the potential of layer-by-layer (LbL) assembly to understand the distance dependence nature of MEF from sulforhodamine B (SRB) assembled on the plasmonic nanostructured surfaces [in the form of Silver Islands films (SIFs)]. The varied proximity of fluorophores from the SIF surfaces was controlled by constructing different numbers of alternate layers of poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH). An anionic laser dye SRB could be electrostatically attached to the positively charged PAH layer. Orientation of the SRB probe molecule adsorbed in PSS/PAH-layered assembly was determined by polarized absorption spectroscopy. The observed tilt angle of the probe transition dipole moment with respect to the surface normal was 40°. Our results show that MEF is indeed distance-dependent. Accordingly, we observed a maximum of a ~6-fold increase in the fluorescence intensity from a monolayer of the SRB at a distance of ~9 nm from the metal-nanostructured surface, with the enhancement decreasing down to ~1.5-fold at about a 30 nm separation distance. Consistently, the minimum lifetimes were about 4-fold shorter than those on glass slides without silver, with the lifetimes being about nearly the same for 15 layers of the PSS/PAH assembly. The intensity-time decays were analyzed with a lifetime distribution model to underpin the distance effect on the metal-fluorophore interaction in the nanometric range. The present study provides improved understanding of the interaction between plasmonic nanostructures and fluorophores and, more importantly, their distance dependence nature, where we used a robust, easy, and inexpensive alternate electrostatic LbL assembly as a bottom-up nanofabrication technique to control the probe distance from the surface.