We report on the build-up and the intrinsic properties of polyelectrolyte multilayer films from poly-L-lysine and glycosaminoglycans (GAGs) with different sulfation degree, i.e. different charge. We used three complementary techniques, namely electrokinetic analysis (EKA), quartz-crystal microbalance with dissipation (QCM-D) and surface plasmon resonance (SPR), to characterize the assembly process and to assess the properties of the obtained films. EKA elucidated the contribution of the polymers charged groups to the net surface charge of the films and suggested that the assembly process is not solely driven by electrostatic interactions. The combined analysis of QCM-D and SPR data demonstrated that the mechanical properties of the films are dependent on the polymer charge: sulfated GAGs (heparin and chondroitin sulfate) form elastic films while hyaluronan (no sulfation) assembles into multilayer constructs with viscous behavior. The contribution of the water content to these distinct regimes is also discussed. Finally, we show that rather complete characterization of the film properties is possible by SPR employing the two-wavelength and two-media approach: thickness, adsorbed mass, refractive index, and interaction kinetics of the assembly process can be studied by SPR alone.