The layer-by-layer deposition method has been used to build a multilayer thin film with two polysaccharides, chitosan CH(weak polycation) and heparin HEP (strong polyanion), on planar quartz surfaces. The film structure and dynamics in aqueous solution were studied with fluorescence resonance energy transfer (FRET) and total internal reflection fluorescence (TIRF). Particular emphasis was placed on the effect of deposition conditions, i.e., pH and salt concentration, on the out-of-plane (vertical) diffusion of fluorescence labeled chitosan in the chitosan/heparin (CH/HEP) film. FRET analysis showed that CH molecules diffused within the film with a diffusion coefficient that was not significantly sensitive to the deposition pH and solution ionic strength. A pH-sensitive label bound to CH embedded within the CH/HEP film was sensitive to the charge of the outermost polymer layer even when buried under 14 alternate layers of CH and HEP. A consideration of the results obtained with both fluorescence techniques showed that the structure of the CH/HEP thin film was highly interpenetrated without clear boundaries between each layer. These results are consistent with the hypothesis that the previously observed exponential-like film growth of CH and HEP in terms of layer thickness and deposited amount versus deposition cycle can be attributed to out-of-plane diffusion of CH molecules in the multilayer.