Reconstruction of extracellular matrix substrates for delivery of functional photoreceptors is crucial in pathologies such as retinal degeneration and age-related macular degeneration. In this study, we assembled polyelectrolyte films using the layer-by-layer deposition method. The buildup of three different films composed of poly(L-lysine)/ chondroitin sulfate (PLL/CSA), poly(L-lysine)/poly(styrenesulfonate) (PLL/PSS), or poly(L-lysine)/hyaluronic acid (PLL/HA) was followed by means of quartz crystal microbalance measurements, optical waveguide light mode spectroscopy, confocal microscopy, and atomic force microscopy. The exponential growth regime and the diffusion of PLL chains from the bulk through the PLL/CSA, PLL/PSS, and PLL/HA films was examined. Evaluation of photoreceptor cell viability was optimal on one layer of PLL (PLL1), followed by 10 bilayers of PLL/HA [(PLL/ HA)(10)] and 10 bilayers of PLL/CSA [(PLL/CSA)(10)]. The number of bilayers and the type of terminating layer also had a significant influence on the number of photoreceptor cells attached. Functionalized polyelectrolyte multilayer films were obtained by adsorbing basic fibroblastic factor (bFGF) or the insoluble fraction of interphotoreceptor matrix (IPM) on or within polyelectrolyte multilayers. bFGF and IPM adsorption on top of the (PLL/CSA)(10)/PLL polyelectrolyte films increased the number of photoreceptor cells attached and maintained the differantiation of rod and cone cells.