This research focused on optimizing the preparations of pDNA-loaded calcium phosphate nanoparticles by employing a 3-factor, 3-level Box-Behnken design. Results indicated that a Ca/P ratio of 189.56, pH of 7.82, and a stirring speed of 528.83 rpm were the optimum conditions for preparation of the nanoparticles. The size of the optimized CaP/pDNA nanoparticles was 61.3±3.64 nm, with a polydispersity index of 0.341 and an encapsulation efficiency of up to 92.11 %. The optimized CaP/pDNA nanoparticles had a high transfection efficiency and demonstrated good biocompatibility in vitro. Therefore, the Box-Behnken design method was successful in providing desirable CaP nanoparticle pDNA delivery systems by optimizing the experimental factors.