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Layer-by-layer polyelectrolyte self-assembly, a common method for preparing high-quality ultra-thin films, was employed to direct the self-assembly behavior of polystyrene-block-poly(methyl methacrylate) (PS-b-PMMA) block copolymer for the first time. Differing from the previous neutral polymer brushes anchored to silicon substrates via chemical modification, polyelectrolyte multilayers (PEMs) were anchored by electrostatic interaction and provided a stable, smooth, and neutral interface. In the present study, PS-b-PMMA was deposited on poly(acrylamide hydrochloride)/poly(acrylic acid) (PAH/PAA) PEMs prepared by layer-by-layer self-assembly to successfully yield vertical nanodomains after thermal annealing. Seven layered PEMs revealed an excellent, smooth surface, with a low roughness of 0.6 nm. The periodic structure with interlamellar spacing of 47 nm was determined by grazing-incidence small-angle X-ray scattering (GISAXS). The morphology of the PS-b-PMMA nanodomains depended on the polyanion-to-polycation concentration ratio, which is related to the interaction between the block copolymer and the substrate. Our results demonstrate that layer-by-layer self-assembly is a helpful method for the phase separation of block polymers and the fabrication of vertical, ordered nanodomains. [ABSTRACT FROM AUTHOR]