Thin films constituted of poly(L-lysine) (PLL) as polycation and of the anionic polysaccharides hyaluronan (HA), chondroitin sulfate (CSA), and heparin (HEP) as polyanions with increasing sulfate contents have been investigated for their internal structure, including water content and ion pairing. Film buildup in physiological solutions was followed in situ by quartz crystal balance with dissipation monitoring (QCM-D) and attenuated total internal reflectance (ATR-FTIR), infrared spectroscopy (ATR-FTIR), which allows an unambiguous quantification of the groups (sulfate, carboxylate, ammonium) present on the side groups of the polyelectrolytes. HA- and CSA-based films were the most hydrated ones. The monomer ratio (disaccharide/lysine) was very similar for all the films, whatever the polyanion, and tended toward a plateau value at approximately 0.5, indicating that there are two lysine molecules per disaccharide monomer. Thanks to the possibility to selectively cross-link carboxylate and ammonium ions via carbodiimide chemistry, the COO-/NH3+ and SO3-/NH3+ ion pairing was determined. We found that 46% of NH3+ groups are unpaired (i.e., extrinsically compensated by counterions) in HA-based films, 21% in CSA-based films and none in HEP ones, which is indeed in agreement with fluorescence recovery after photobleaching (FRAP) measurements of fluorescently labeled PLL diffusion in the films. In addition, the ratio of SO3- versus COO- pairing with NH3+ groups was close to the stoechiometry of these groups in the dissacharide monomeric unit, that is, 2:1 for HEP-based films and 1:1 for CSA based films. Thus, hydration, ion pairing, and PLL diffusion in the films are interconnected properties that arise from the specific structures of the biomacromolecules constituting the films.