The objective of this study was to develop a novel three-dimensional biomimetic gelatin/multiwalled carbon nanotubes/hydroxyapatite (gelatin/MWNTs/HA) nanofibrous scaffold via electrospinning technique for bone tissue engineering. The mechanical properties, structure, morphology and the bioactivity of nanofibrous scaffolds in vitro were investigated. Attentions were focused on the adhesion, mineralization, viability and proliferation of human fetal osteoblastic cells (hFOBs) on scaffold. Resulting scaffolds provided relative good mechanical support (7.9 +/- 0.32 MPa) and high porosity (91.2%) to mimic a favorable environment for hFOBs. The hydrogen bonds between gelatin molecules and MWNTs/HA units were confirmed, and the incorporation of HA or MWNTs/HA nanoparticles caused an increase in porosity and strength of scaffolds, meanwhile the surface of nanofibers tended to be rough. HA nanoparticles showed a chelating effect to promote osteogenesis and mineralization of bone, and MWNTs had a synergetic effect with HA to induce the apatite formation. As compared to gelatin and gelatin/HA scaffolds, gelatin/MWNTs/HA scaffold exhibited the best viability hFOB cells cultured in vitro, most excellent morphology of hFOB cells seeded into scaffold and a significantly increasing in proliferation. The nanofibrous scaffold will have great potential as an excellent scaffold for in bone tissue engineering.