Nanotechnology plays a unique and novel role to develop new methods for genetic engineering. Calcium phosphate (CaP) has various significances in biomedical systems; one of them is plasmid DNA (pDNA) delivery for decades as transfection efficiency relative to the non-viral approaches. In this study, we developed a novel gene transfer carriers, nano-calcium-phosphate (CaP) that provides consistently efficient and satisfactory pDNA delivery in tobacco. Results showed that CaP nanoparticles successfully delivered pBI121 harboring GFP driven by 35S promoter-encoding plasmid DNA into tobacco cells. Characterization of nanoparticles were done using DLS, TEM and found 25-55 nm range. While X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) analyses were done to confirm the hydroxyapatite nature of the synthesized CaP nanoparticles. Polymerase chain reaction (PCR) detection, were carried out for the regenerated plants. The established genetically transformed tobacco plants mediated by CaP nanoparticles can be achieved. These studies revealed the significant efficacy of CaP nanoparticles as non-viral gene delivery in tobacco plant transformation and proved a novel system for plant genetic modification.