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Electrostatic layer-by-layer (LBL) self-assembly is an attractive method for depositing films, composed of charged molecules, on a wide variety of charged substrates. This report describes progress toward development of LBL as a platform for fabrication of fluorescent sensors based on nanocomposite multilayer ultrathin films. This study had two main features: 1) assessment of methods for immobilizing indicators using LBL and 2) demonstration of the versatility of these methods for application to templates of specific interest for optical sensing. Ruthenium-based oxygen indicators were used as model fluorophores. Three techniques for building fluorescent sensing films were considered, including direct electrostatic assembly of charged fluorescent indicators, fluorophore:polyion premixing, and conjugation of indicator to polyelectrolyte. Dye-conjugated polyion films exhibit superior linearity in growth, stability, and resistance to desorption during the assembly process. Multilayer fluorescent films containing bis(2, 2'-bipyridine) 4'-methyl-4-carboxybipyridine-ruthenium-N-succinimidyl-ester bis(hexafluoro-phosphate) (Ru(bpy)2(mcbpy)) conjugated to poly(allylamine hydrochloride) (PAH) were deposited onto glass slides, fiber-optics and polymer microspheres. Additional layers of fluorescein 5(6)-isothiocyanate (FITC) conjugated to PAH were deposited on the same templates to serve as an internal reference and allow ratiometric measurements. The fluorescence properties of films on the different substrates were similar, demonstrating that the deposition process is versatile and generally portable between templates. In all cases, the films retained oxygen sensitivity and did not exhibit significant self quenching. This work provides a basis for development of sensors for numerous biomedical sensing applications, including fiber-optic probes designed for research and clinical measurements, sensing films on tissue culture substrates, and implantable micro/nanoparticle based sensors for in vitro or in vivo monitoring.