In this paper, polyethylenimine (PEI) and Chitosan were simultaneously one-step doped into silicon dioxide (SiO ) nanoparticles to synthesize PEI/Chitosan/SiO composite nanoparticles. The polymer PEI contained a large amount of amino groups, which can realize the amino functionalized SiO nanoparticles. And, the good pore forming effect of Chitosan was introduced into SiO nanoparticles, and the resulting composite nanoparticles also had a porous structure. In pH 7.4 phosphate buffer solution (PBS), the amino groups of PEI had positive charges, and therefore the fluorescein sodium dye molecule can be loaded into the channels of PEI/Chitosan/SiO composite nanoparticles by electrostatic adsorption. Furthermore, utilizing the diversity of DNA molecular conformation, we designed a high sensitive controllable assembly of DNA gated fluorescent sensor based on PEI/Chitosan/SiO composite nanoparticles as loading materials. The factors affecting the sensing performance of the sensor were investigated, and the sensing mechanism was also further studied.