We demonstrate self-assembly of fluorescent polymer nanospheres on a nanostructured azobenzene molecular thin film, and evaluate optical near-field excitation of aligned nanospheres. Nanogratings on azo thin film optically fabricated utilizing standing evanescent waves were used for an alignment layer of nanospheres. After dropping and air-drying a solution of nanospheres, the nanospheres were aligned along the grooves of nanograting. The variations in distribution of nanospheres were observed for a choice of grating shape and dispersing solvent of nanospheres. The size ordered alignment of nanospheres was found by using spin coating. To evaluate the fundamental processes of optical near-field excitation of the nanospheres, the emission light from the fluorescent nanospheres periodically aligned on azo grating was observed. The angular distribution of emission lights at a prism side of the angles larger than critical angle was confirmed, which correspond to the near-field optical excitation mediated by evanescent waves radiated near nanospheres.