We report in this paper the preparation of multilayer Films from the layer-by-layer deposition of block copolymer nanofibers bearing carboxyl and amine groups in their coronas. The film formation process was followed by atomic force microscopy and UV absorbance measurements. For the large size of the nanofibers and probably also for the difficulty associated with reshuffling the positions of nanofibers once they were adsorbed by an underlying layer, nanofibers could not be packed densely. We took advantage of this "defective" feature and showed that these porous nanofiber multilayer films could be used to separate nanospheres of different sizes and probably also of different surface functionalities.