The Forster resonance energy transfer (FRET) between 6-carboxyfluorescein (6-CF) (donor) and rhodamine B-labeled melamine formaldehyde (RhB-MF) particles (acceptor) in aqueous solution was exploited to investigate the layer properties of polyelectrolyte (PE) multilayers preadsorbed on the particle surface. The formation of poly(styrenesulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) multilayers on the RhB-MF particles was confirmed by electrophoretic mobility measurements. The FRET process was found to proceed via adsorption of 6-CF onto the RhB-MF particles and was thus dependent on the degree of surface coverage of the PE on the surface. The PE surface coverage could be altered by depositing the layers with or without added electrolyte. The extent of FRET was also influenced by the number of PE layers (and hence layer thickness) surrounding the RhB-MF particles. Increasing the number of PE layers resulted in less energy transfer, reflecting less accessible sites on the RhB-MF particles for 6-CF adsorption. The PE layers were found to be permeable to 6-CF, with no diffusion effects evident on the time scale of the steady-state fluorescence measurements. Further, 6-CF was found to interact with PAH when the outer PE layer on the particles was PAH. This interaction presents a novel way of detecting amino sites of PAH not interacting with PSS in the PE multilayer films.