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TiO2/PSS nano-structured multilayer films are fabricated by a layer-by-layer self-assembly method, and the deposition process and interface structure of films are studied in detail by slow positron Spectroscopy. The results indicate that injection energy of positron at the interface between the substrate and the film shows a linear dependence on the number of bilayer, which suggests that the repeatability of the depositing process is good, and the thickness of films shows a linear increase with the number of bilayers adding. The calculated result of the film thickness shows that there is an overlay between the adjacent TiO2 nanoparticle layers.