The design of advanced, nanostructured materials at the molecular level is of tremendous interest for the scientific community because of their potential in several fields, including medicine, biology and tissue engineering. Layer-by-layer (LbL) assembly is a versatile technique for the realization of multilayered films with tailored characteristics at the nanometer scale. Photoactive nanostructured films were prepared by LbL assembly of photozymes: an amphiphilic zwitterionic copolymer, poly(sodium styrene sulphonate-stat-vinyl naphtalene-stat-3dimethyl(methacryloylethyl) ammonium propane sodium sulfonate) (PSSS-stat-VN-stat-DMPAS; code: ZI), and a cationic polyelectrolyte, chitosan-g-fluorescein (code: CHFL), on a crosslinked gelatin substrate. Although successful LbL assembly of photozymes was proven by the performed characterization, gelatin was not an optimal substrate for the coating, due to its low charge density and amphoteric nature. A regular growth of the layers was found after at least 6 layers were deposited. The macromolecules of the zwitterionic photozyme adopted a coiled micellar conformation in solution which was kept during the assembly. The obtained nanostructured films are promising candidates for carrying out efficient electron transfer process within them, responsible for the anti-microbial activity and the osteointegration ability of the coating.