Fluorescent polymeric spheres with uniform sizes are of great interests in the measurement and calibration of the pore sizes and defects of porous media, including separating membranes. We reported the preparation of such nanospheres by dissolving amphiphilic block copolymers of polystyrene-block-poly(2-vinyl pyridine) (PS-b-P2VP) together with chromophores (perylene and pyrene) in acetic acid at elevated temperatures. Micellar nanosphere with the chromophores residing in the cores were formed through the heating-enabled micellization process. Following quaternization of the P2VP coronae gave the spheres a positively charged surface and a good water dispersibility. Scanning and transmission electron microscopy and dynamic light scattering analysis confirmed that the micellar nanospheres were uniform in size both in the dry and wet state. The micelle size and the thickness of coronae can be tuned by altering the molecular weight of the constituent blocks. Encapsulation of chromophores in the micelle cores provided a strong fluorescent emission to the micellar nanospheres, while quaternization of the P2VP coronae gave the nanospheres a greatly variable size according to pHs and salt concentrations in the surroundings. Taking advantages of their uniform size, water dispersibility, and fluorescent emission for accurate detection, we demonstrated that the micellar nanospheres were capable of characterizing pore sizes of separating membranes and detecting their large-pore defects. (C) 2013 Elsevier BY. All rights reserved.