In the present study, we had introduced polyurethane (PU) nanofibers that contain hydroxyapatite (HAp) nanoparticles (NPs) as a result of an electrospinning process. A simple method that does not depend on additional foreign chemicals had been employed to synthesize HAp NPs through the calcination of bovine bones. Typically, a colloidal gel consisting of HAp/PU had been electrospun to form nanofibers. In this communication, physiochemical aspects of prepared nanofibers were characterized by FE-SEM, TEM and TEM-EDS, which confirmed that nanofibers were well-oriented and good dispersion of HAp NPs, over the prepared nanofibers. Parameters, affecting the utilization of the prepared nanofibers in various nano-biotechnological fields have been studied; for instance, the bioactivity of the produced nanofiber mats was investigated while incubating in simulated body fluid (SBF). The results from incubation of nanofibers, indicated that incorporation of HAp strongly activates the precipitation of the apatite-like particles, because of the HAp NPs act as seed, that accelerate crystallization of the biological HAp from the utilized SBF.