Bone Tissue Engineering (BTE) composed of three main parts: scaffold, cells and signaling factors. Several materials and composites are suggested as a scaffold for BTE. Biocompatibility is one of the most important property of a BTE scaffold. In this work synthesis of a novel nanocomposite including layered double hydroxides (LDH) and gelatin is carried out and its biological properties were studied. The co-precipitation (pH=11) method was used to prepare the LDH powder, using calcium nitrate, Magesium nitrate and aluminum nitrate salts as starting materials. The resulted precipitates were dried. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) analyses were used to characterize the synthesized powders. The results demonstrated the presence of nanocrystals of Ca-LDH and Mg-LDH as Hexagonal and Layered Morphology. The obtained powders were composed to gelatin via solvent casting method then freez dried. The scaffold was prepared via membrane lamination method from the resulted layers that linked together with gelatin as binder. In order to investigate the scaffold cytotoxicity MTT assay was done with a osteosarcoma cell line. No toxic response was observed in specimens. As a major result, it was demonstrated that the specimen showed a significant cellular response. Then osteosarcoma cells were cultured for 7-day and 14-day extract of powders. The composites osteoconductivity was investigate with cells alkaline phosphatase extraction. The results demonstrated that the Ca-LDH/gelatin composite scaffold has a good potential for bone tissue engineering applications and Mg-LDH specimen has a better osteconductivity.