The separation of target substances is a signi cant biological detection procedure, where magnetic microspheres can act as high-performance separation materials. However, challenges are still kept to ful ll all the requirements. In this study, a type of submicron magnetic poly (glycidyl methacrylate) (PGMA) microsphere was prepared with an in situ coprecipitation method, an electrostatic self-assembly method, and a silica surface coating method. Firstly, the PGMA microspheres were synthesized by a soap-free emulsion polymerization method, and surface charge density determined the coagulation process, further in uencing the size and monodispersity. Then we found the Superparamagnetism properties of magnetic microspheres could be well controlled by the capping agent sodium citrate (Na 3 Cit), and the superparamagnetic critical size was 10.9 nm. Also, the saturation magnetization was well controlled by the Fe 2+ and Fe 3+ concentration, which was correlated with the nucleation rate of Fe 3 O 4 crystal. Furthermore, we proved that the electrostatic self-assembly was guided by pH, and it was proposed to tightly couple the PGMA-NH 2 microspheres with positive charges and Fe 3 O 4 nanoparticles with negative charges. Finally, the PGMA@Fe 3 O 4 microspheres were coated with SiO 2 , surface modi ed by carboxyl groups for application. The PGMA@Fe 3 O 4 and carboxyl-modi ed microspheres exhibited saturated magnetization values of 23.73 and 17.73 emu/g, respectively. These microspheres have been effectively utilized for the extraction of DNA from various sources such as Salmonella typhi, monkeypox virus, and clinical swab samples, suggesting the potential of these microspheres for nucleic acid separation in the biomedical domain.