In this study, a fibrous nanocomposite scaffold was developed by combining hydroxyapatite (HA) fibers produced by electrospinning method and arginine-glycine-aspartic acid (RGD)-bearing peptide-amphiphile (PA) gel (PA-RGD) produced by self-assembly and gelation induced by calcium ions. Scanning electron microscope, transmission electron microscope and atomic force microscopy imaging confirmed the successful production of inorganic and organic components of this nanocomposite material. Within the HA, the presence of a CaCO3 phase, improving biodegradation, was shown by x-ray diffraction analysis. The in vitro effectiveness of the PA-RGD/HA scaffold was determined on MC3T3-E1 preosteoblast cultures in comparison with HA matrix and PA-RGD gel. The highest cellular proliferation was obtained on PA-RGD gel, however, alkaline phosphatase activity results denoted that osteogenic differentiation of the cells is more favorable on HA containing matrices with respect to PA-RGD itself. Microscopic observations revealed that all three matrices support cell attachment and proliferation. Moreover, cells form bridges between the HA and PA-RGD components of the nanocomposite scaffold, indicating the integrity of the biphasic components. According to the real time-polymerase chain reaction (RT-PCR) analyses, MC3T3-E1 cells expressed significantly higher osteocalcin on all matrices. Bone sialoprotein (BSP) expression level is ten-fold higher on PA-RGD/HA nanocomposite scaffolds than that of HA and PA-RGD scaffolds and the elevated expression of BSP on PA-RGD/HA nanocomposite scaffolds suggested higher mineralized matrix on this novel scaffold. Based on the results obtained in this study, the combination of HA nanofibers and PA-RGD gel takes advantage of good structural integrity during the cell culture, besides the osteoinductive and osteoconductive properties of the nanofibrous scaffold.