Protein and polysaccharide nano-patterning is of prime interest for biological applications but also for applications in the field of diffractive optics. In this work, we used a photo-nano-patterning process based on light interferences through a photo-sensitive material for patterning polysaccharides and polypeptides pure and mixed gels of gelatin, hyaluronan, and chitosan. Chromium ions were incorporated in the gels to render them photo-sensitive. Polyelectrolyte multilayer thin films of poly(L-lysine)/hyaluronan were also investigated either by incorporating chromium ions or by adsorbing a photo-sensitive hyaluronan. Depending on the weights ratios of the polymers, respectively gelatin/chitosan and gelatin/hyaluronan, the gel surfaces exhibit different fringe patterns, as can be visualized by atomic force microscopy. The diffracted intensity characterizing the holographic grating was also depending on gel type. Pure gelatin gels was taken as the reference material. The best results in terms of surface patterns and diffracted intensities were obtained for the gelatin/chitosan gels prepared at acidic pH and exposed at energies ranging from 100 to 400 mJ/cm2. Our results show that surface patterns of various depths and structures can be created by the photo-patterning technique on biological polymers. These results open new perspectives for the surface control of biological materials but also for making use of the optical properties of these biocompatible biopolymers.