Orientation of azo chromophores in electrostatic self-assembled multilayer films of four side-chain azo polyelectrolytes that contain azobenzene derivatives with different p-substituents was investigated by using polarized UV-Vis absorption spectroscopy.Influences of substituents on the orientation of the azo chromophores were discussed in detail.The structural characteristics of these self-assembled films were further investigated.It was found that the homogeneous orientation tendency of azobenzene was a common phenomenon in the self-assembled films fabricated by electrostatic layer-by-layer dipping process of the side-chain azo polyelectrolytes and poly(diallyldimethyl ammonium chloride); substituents that link to azobenzene have influences on the orientation of azobenzene in self-assembled films,which could be attributed to charge interaction and dipolar interaction between the chromophores and the polycation substrates.For those self-assembled multilayers with H-aggregation, the effect of substituents on the orientation of azobenzene was also influenced by the H-aggregation.The results indicate that the orientation of azobenzene in self-assembled films can be effectively adjusted by selecting polyelectrolytes containing azobenzenes with different p-substituents.