Improving the detection sensitivity of enzyme linked immunosorbent assay (ELISA) is of the utmost importance for meeting the demand of early disease diagnosis. In this work, a sensitive solid substrate for ELISA, i.e., hierarchical iron oxide magnetic microspheres, Fe₃O₄@mSiO₂@poly[poly(ethylene glycol) methacrylate-co-glycidyl methacrylate], was developed via a novel surface-initiated photoiniferter-mediated polymerization (SI-PIMP) strategy. The magnetic microspheres consist of a magnetic Fe₃O₄ core that gives a high magnetic response, a 3D backbone, a mesoporous SiO₂ middle layer, that facilitates microsphere stability and provides anchoring sites, and polymer brushes, that serve as an antifouling and oriented antibody immobilization layer. As a result, the as-prepared microspheres possess a high antibody loading capacity, an enhanced detection signal and a dramatically improved sensitivity, resulting in a 25-fold improvement over conventional ELISA solid substrates.