The use of materials based on protein-polymer compositions is a promising method for solving a number of problems in biotechnology and medicine. In our work, we produced multilayer nanofilms based on 4th generation (G4) pyridylphenylene dendrimers with fully pyridine-based periphery containing a protein component. For dairy beta-and kappa-caseins, alpha-lactalbumin, recombinant sheep prion protein, lysozyme, trypsin, and alpha-chymotrypsin the ability to integrate into self-organizing structures about 230-710 nm thick, measured by atomic force microscopy, was found. The formation of nanofilms is dependent on the protein/dendrimer ratio in the initial solution. At the same time, the nanofilms are resistant to pH changes, and the action of detergents. We demonstrated that trypsin and chymotrypsin incorporated into the nanofilms still conserved proteolytic activities and were stable for at least 3 weeks. We have also conducted in silico molecular modeling of the G4 dendrimer interactions with lysozyme. Spatial structure of the dendrimer-protein complex was predicted and most probable variant remained almost intact under 100 ns molecular dynamics simulation conditions. We believe that dendrimer-protein systems are promising tools for possible applications in the development of new stable bio-materials and biosensors. (c) 2018 Elsevier Ltd. All rights reserved.