The rapid purification of biomaterials such as DNA, RNA, and antibodies has attracted extensive attention, and research interest has increased further with the COVID-19 pandemic. In particular, core–shell-structured superparamagnetic nanoparticles have been continuously studied for their application as biopurification materials. It has been reported that Fe3O4@SiO2 nanoparticles are one of the most promising candidates for separating nucleic acids via a simple and rapid process. This study proposed a fabrication method for dual-layered Fe3O4@SiO2 nanoparticles, in which the density of the SiO2 shell was controlled using an intermediate surfactant during the SiO2 coating. After the fabrication of dual-layered Fe3O4@SiO2 nanoparticles, structural, morphological, and magnetic analyses were conducted. The results showed that the Fe3O4 nanoparticles were surrounded by a dense layer 15.6~27.9 nm thick and a porous layer 24.2~44.4 nm thick, and had superparamagnetic properties with high saturated magnetization at room temperature (86.9 emu/g). Then, the optimal conditions for the biopurification material were suggested based on analysis of the selective separation of plasmid DNA.