We deposited homogeneous, thin, yellow-colored films of chitosan (Chi)-N,N-Dimethyl-4-(2-pyridylazo)aniline (PADA) dye from an acid Chi–PADA solution by spin-coating on glass substrates. We characterized Chi, PADA, and Chi–PADA films by ATR–FTIR spectroscopy, which revealed a slight shift of 3170 and 3268 cm−1 bands, indicating H-bonding between the chitosan hydroxyl (OH) group and the amine (N) of the PADA pyridine ring. Based on these analyses, it was possible to determine the efficiency of the hydrogen bonds to form a Surface Relief Grating (SRG) on azo-polymer thin film. Moreover, we performed UV–VIS spectroscopy analysis of this film, which showed a broad band extending from 400 to 700 nm, with the maximum occurring at 428 nm. Therefore, we selected, within the absorption band, the 532 nm green laser wavelength to irradiate the azo-polymer films at room temperature. For the first time, natural polymer derivative and dye sample Chi–PADA thin films showed unique photoresponsive behavior under irradiation with two interfering laser beams. This permitted us to generate surface inscription patterning known as an SRG, which we confirmed by atomic force microscopy (AFM) and for which we determined a grating depth up to 50 nm. The present study opens the new possibility of using natural polymer-dye thin films.