The novel hierarchical microspheres from conjugated poly(p-phenylene)s can be self-assembled by the non-solvent vapor method. The formation of hierarchical microspheres depends on the choice of polar nonsolvent vapor atmospheres, solvents, and the length of side chains. The intermolecular interactions, including the side-chain interactions and the pi-pi interactions between conjugated backbones, occur spontaneously to form various conformations and polymeric aggregates, which have an effect on the fluorescence wavelength of the polymer hierarchical microspheres. Our results demonstrate a facile way to prepare polymeric hierarchical microspheres and adjust their optical properties. Moreover, the hierarchical microspheres with rough surface show a highly selective fluorescence "turn-off' sensor for Fe3+ with almost no interference of other metal ions.