Aims: This study aimed to prepare and physicochemically evaluate as well as assess the cytotoxicity and stimulation of early osteogenic differentiation of dental pulp stem cells of gelatin-hydroxyapatite (Gel-HA) fibrous nanocomposite scaffold. Background: Recently, the electrospinning approach in nanotechnology has been considered due to its application in the preparation of biomimetic nanofibers for tissue engineering. Objective: The main objective of this study was to evaluate Gel-HA fibrous nanocomposite for regenerative dentistry and bone tissue engineering material. Methods: The nano-scaffold was prepared via the electrospinning method. Then, the physicochemical properties (particle size, surface charge, morphology, hydrophilicity, specific surface area, crystalline state and the characterization of functional groups) and the proliferative effects of nano-scaffolds on dental pulp stem cells were assessed. The alkaline phosphatase activity was assessed for evaluation of early osteogenic differentiation of dental pulp stem cells. Results: The prepared nano-scaffolds had a negative surface charge (-30 mv±1.3), mono-dispersed nano-scale diameter (98 nm±1.2), crystalline state and fibrous uniform morphology without any bead (structural defects). The nanofibrous scaffold showed increased hydrophobicity compared to gelatin nanofibers. Based on Brunauer-Emmett-Teller analysis, the specific surface area, pore volume and pore diameter of Gel-HA nanofibers decreased compared to gelatin nanofibers. The Gel-HA nano-fibers showed the proliferative effect and increased the alkaline phosphatase activity of cells significantly (P<0.05). Conclusion: The prepared Gel-HA nanofibers can be considered potential candidates for application in bone tissue engineering and regenerative dentistry. Other: Gel-HA nanofibers could be a potential material for bone regeneration and regenerative dentistry in the near future.