Polyurethane films used in wound dressings often have good traarency, flexibility, blood compatibility and permeability to water vapor and oxygen, but surface modifications must be made to enhance biocompatibility. For this purpose, traarent films prepared from polyurethane synthesized from castor oil and Desmodur® N75, after assuring complete polymerization reaction by ATR-FTIR spectra results, polyurethane pieces in dimensions 2×2 cm 2 were treated with low pressure nitrogen plasma, at pressure of 0.2 mbar and power of 60 W. By measuring the static contact angle of water at different times during the plasma process, which was directly related to the amount of peroxide formed, also the use of two types of oxygen and nitrogen gas, optimized plasma process conditions were investigated. The lowest contact angle was related to the use of nitrogen gas after 120 s. The films after undergoing the plasma process and forming peroxide groups on the surface, immersed in 10%, 30%, 50%, 70% and 90% (w/v) acrylic acid solution, then grafted onto the surface by polymerization reaction at 80?C in an oven. Based on the results of ATR-FTIR, SEM images and contact angle measurements, acrylic acid solution with 70% concentration was considered for bonding with the polyurethane film surfaces. Acrylic acid grafted films were first activated by EDC/NHS solution and then reacted with collagen and chitosan biomolecules separately. The results of ATR-FTIR, contact angle measurements, SEM and AFM images, qualitative and quantitative results of fibroblast cell culture and antibacterial properties of untreated polyurethane film compared with modified polyurethane films were evaluated. The results demonstrated that collagen and chitosan-modified films increased the growth and proliferation of fibroblast cells. Chitosan immobilized film showed stronger antibacterial properties than collagen immobilized film, therefore, in layer by layer self-assembly of biomolecules on the film surface, chitosan was considered as the outermost layer. In the second part of this study, collagen and chitosan as polycationic, as polyanionic between each of the two biomolecule layers were deposited on the polyurethane film surface by performing the layer by layer self-assembly, in three and five steps layer deposition. Prior to the layering process, the film surface was first modified by three different methods, A, B and C. In Method A, the film grafted with polyacrylic acid was converted to a negatively charged carboxylate ion by immersing in NaOH solution, in method B, polyacrylic acid grafted film was first reacted with collagen and then immersed in solution containing negative charge. In method C, the film grafted with polyacrylic acid, after reaction with hexamethylene diamine was aminolized and by immersion in solution contained negative charge. Before the LbL self-assembly process, in the first section of this, the collagen and chitosan biomolecules were stained by quantum dots, and in the second section, biomolecule staining was performed with fluorescent material. The results of FESEM, confocal microscopy images, AFM and ellipsometry tests showed that the best method was related to LbL self-assembly process when the film surface was aminolized in method C and more layers of chitosan and collagen biomolecules had been deposited. However, greater presence of these biomolecules increased the rate of growth and proliferation of fibroblast cells, but notable point is that in all three methods used in the LbL self-assembly process, the three-layer mode exhibited better cell proliferation and cell viability than the five-layer mode. In terms of antibacterial properties, generally films containing more layers of collagen and chitosan showed less resistance to Gram-positive and Gram-negative bacteria. therefore, surface modification of polyurethane film by performing LbL self-assembly of collagen and chitosan biomolecules, increased the cytocompatibility of the film even compared to the film coated with the monolayer of these biomolecules, but in terms of antibacterial properties it was better only than untreated polyurethane film. Keywords: Polyurethane, Low pressure plasma treatment, Collagen, Chitosan, Sodium polystyrene sulfonate, Layer-by-Layer self-assembly