High-performance composites containing various kinds of nanofibers as reinforcing building blocks have recently received considerable attention, owing to their superior mechanical properties. One of the effective strategies to reinforce these composites involves strengthening interfacial interactions via covalent bonds. However, in contrast to nanosheets, covalent bonds have been rarely used in nanofiber-reinforced composites. Herein, we report the macroscale fabrication of a series of Ag nanowire (NW)-thiolated chitosan (TCS) composite films via spray induced self-assembly. The obtained films were significantly strengthened by Ag-S covalent bonds formed between the Ag NWs and the thiol groups of TCS. The tensile strength of the optimized Ag NW-TCS film was up to 3.9 and 1.5 times higher compared with that of pure TCS and Ag NW-chitosan (CS) films, respectively.