Highly responsive monodisperse microsized magnetic silica microspheres were synthesized through the electrostatic self-assembly method. Monodisperse composite silica microspheres were prepared by depositing tetraethyl orthosilicate prepolymer on the glycidyl methacrylate-co-ethylene glycol dimethacrylate microspheres. After calcination, the organic moiety was removed and macroporous microspheres were left on the silica particles. To increase the selectivity, magnetic porous silica was bonded with different alkyl functional groups. The morphology, particle size distribution, magnetic properties, and functional groups of magnetic porous silica microspheres were characterized by scanning electron microscopy, coulter counter, vibrating sample magnetometry, X-ray diffraction, and elemental analysis, respectively. Extraction parameters, including pH, the salt concentration of the adsorbed solution, adsorption time, elution solvent, and time, were systematically investigated. The developed magnetic solid-phase extraction method, combined with high-performance liquid chromatography, presented reasonable linearity (0.5-10 µg/mL), low limit of detection (45.0-108.0 ng/mL), and high selectivity for cefadroxil, cefquinome, and cefradine. It was operated in three kinds of cephalosporins detection from wastewater with the relative recovery rates in the range of 86.5-97.0%. These results indicated that the synthesized magnetic microsphere is a promising adsorbent for the enrichment of cephalosporins.