In recent years, nanoparticle systems have been frequently used as non-viral carriers for the treatment of genetically based diseases and cancer. However, the conjugate formation should be optimized by selecting a suitable carrier for the transport of each genetic material desired to be transported to the target cell. A suitable conjugate should be created by taking into account the size and zeta potential electrical characteristics of the cargo being transported. Nanocarrier systems can protect and package genetic material. Calcium phosphate nanoparticles (CaP NPs) with unique advantages have been a promising approach for in vitro and in vivo gene transfers. However, the biggest challenge of the CaP NPs is their unreducible size. In this study, CaP NPs were synthesized via the co-precipitation method using two stabilizing agents separately: magnesium nitrate and sodium citrate. The transfection efficiency of nanoparticles obtained by using sodium citrate in the MCF-7 cell line was observed to be more effective compared to magnesium nitrate-containing nanoparticles. The nanoparticles with a size of (248.1 ± 54) nm (polydispersity index (PDI), 0.3) and zeta potential of (–13.3 ± 2.8) mV were synthesized. The morphological properties of the particles were determined by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Cytotoxicity of nanoparticles was performed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis using L929 mouse fibroblast cells. In the study, both CaP/plasmid DNA conjugation and naked plasmid DNA were transferred to MCF-7 cells, and the gene expression efficiency was determined by fluorescent microscopy via green fluorescent protein (GFP) expression. Gene expression efficiencies were determined as 24% (±1.7%) for naked pDNA and 76% (±2.3%) for CaP-conjugated pDNA, respectively.