In this article, we present an easy route to prepare monodisperse core-shell Fe3O4@SiO2 microspheres with uniform size and shape. Their structures and properties were studied by transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared spectrometry (FT-IR), and vibrating sample magnetometer (VSM), respectively. The results showed that spherical Fe3O4 microspheres with well dispersion have a rough surface and an average diameter (about 500 nm). After the modification with silica, the particles have a well-defined core-shell structure and a much smoother surface and larger particle diameter (about 600 nm). Furthermore, VSM measurements indicated that the as-prepared Fe3O4 and Fe3O4@SiO2 microspheres were superparamagnetic at room temperature and the saturation magnetization (M(s)) were 58.110 emu/g and 33.479 emu/g, respectively. And then, the prepared monodisperse core-shell Fe3O4@SiO2 microspheres were subsequently applied to separate nucleic acids from the bacteria (E. coli BL21) and verified the great application prospects for bioseparation technology of the biomoleculars.