In this study, polyurethane (TPU) scaffolds were fabricated using freeze-drying technique and gelatin macromolecules immobilized on the activated surface by oxygen plasma treatment. Scanning electron microscopy (SEM) micrographs indicated an interconnected porous microstructure with randomly oriented pores. According to the results, the diameter of pores increased after plasma treatment and gelatin grafting. Fourier transform infrared spectroscopy illustrated that there is no inappropriate interaction between materials during processing; furthermore, attenuated total reflection Fourier transform infrared spectroscopy confirmed the immobilization of gelatin molecules on the surface of the plasma-treated polymeric scaffolds. Waterdrop contact angle analysis presented that wettability and hydrophilicity of constructs increased after grafting gelatin on the activated surface. Phosphate-buffered saline absorption and hydrolytic biodegradation enhanced after surface modification of the polymeric samples. Cellular behavior demonstrated better adhesion and spreading after grafting gelatin of oxygen plasma-treated constructs. No evidence of toxicity was observed for 7 days. DNA content determined that the number of viable cells increased in TPU-gelatin matrixes after 1 day in contrast with TPU scaffolds. Based on results, oxygen plasma treatment can create an activated surface to graft gelatin macromolecules and achieve optimum physicochemical, mechanical, and biological features for the neo-tissue formation.