Tissue engineering scaffolds simulate extracellular matrixes (ECMs) to promote healing processes of damaged tissues. In this investigation, ECM were simulated by retinoic acid-loaded polyurethane-graphene oxide nanofibers to regenerate bone defects. Scanning electron microscopy (SEM) micrographs, Fourier transform infrared (FTIR) spectrum and X-ray diffraction (XRD) patterns proved the synthesis of graphene oxide (GO) nanosheets. SEM micrographs of nanofibers demonstrated through the formation of homogeneous and bead free fibrous scaffolds that the diameter of fibers were reduced by decreasing the applied voltage in an electrospinning process and the addition of GO. According to the results, the addition of GO to the polyurethane (PU) solution led to an increase in mechanical strength which is the most important parameter in the hard tissue repair. The GO-containing scaffolds showed an increased wettability, swelling, biodegradation and drug release level. Release behavior in nanocomposite scaffolds followed the swelling and biodegradation mechanisms, so osteogenic expression was possible by incorporating retinoic acid (RA) in PU-GO nanofibrous scaffolds. Biological evaluations demonstrated that composite scaffolds are biocompatible and support cellular attachment in which RA-loaded samples represented better cellular spreading. In brief, nanocomposite fibers showed desired that the physicochemical, mechanical and biological properties and synergic effects of GO and RA in osteogenic activity of MG-63 cells produced favorable constructs for hard tissue engineering applications.