Two sulfonated poly(sulphone) sodium salts (SPSF) with different molecular weights and ionic exchange capacities in a N,N-dimethyl formamide/water (DMF-H2O) mixed solvent with various DMF contents were selected as a model system for investigating the influences of solvent composition, solution properties, and charge density of polyelectrolytes on the layer-by-layer (LbL) self-assembly of water-insoluble polyelectrolytes. Poly(dimethyldiallylammonium chloride) (PDDA) in aqueous solution was used as the counterpart. The PDDA/SPSF multilayer films grew nearly linearly with the layer numbers regardless of the volume fraction of DMF, phi(DMF), in the SPSF solutions. The total absorption amount of the PDDA/SPSF multilayer films was strongly dependent on the charge density of the SPSF molecules and the phi(DMF) value of the SPSF solutions. Minimum values of absorption amount were observed at phi(DMF) = 0.6 to similar to 0.7. The surface hydrophobicity and roughness of the multilayer films can be tuned by varying phi(DMF). These observations were rationalized in terms of the chain dimension and the ionization degree of the SPSF molecules as a function of phi(DMF), which was characterized by the intrinsic viscosity ([eta](SPSF)) and conductivity (L-SPSF) of the SPSF solutions. The results indicate that the molecular structures of the DMF-H2O mixed solvent strongly affect the solution properties of SPSF, which in turn determine the growth behavior and physical properties of the PDDA/SPSF multilayer films.