The Fe3O4@SiO2@vmSiO2 microspheres with ordered mesochannels and large inter-lamellar void were successfully prepared through stepwise solution-phase interface deposition. Fe3O4 nanoparticles were coated with SiO2 via the Stöber method, and they were further coated with mesoporous SiO2 using aggregation of cetyltrimethylammonium chloride as template to prepare Fe3O4@SiO2@vmSiO2. The Fe3O4@SiO2@vmSiO2 microspheres show a well-defined core–shell structure with high magnetization (~ 30.9 emu g−1), ordered mesochannel (~ 6.8 nm in diameter), and inter-lamellar void (~ 30 nm). Laccase (LAC) was immobilized on a modified Fe3O4@SiO2@vmSiO2 microsphere by covalent attachment and stabilized onto the glassy carbon electrode (GCE) surface (Fe3O4@SiO2@vmSiO2-LAC/GCE) in the fabrication of novel immobilized LAC biosensors for monitoring dopamine (DA). The electrochemical properties of the biosensor were investigated with electrochemical impedance spectroscopy and cyclic voltammetry. The immobilized LAC biosensor possesses good DA electrocatalytic activity with a linear range of 1.5–75 μmol L−1 and low detection limit of 0.177 μmol L−1 and shows strong anti-interference ability and excellent selective determination of DA that coexists with ascorbic acid. The immobilized LAC biosensor was also used to detect DA in pharmaceutical injection. The recoveries of 98.7–100.5% were obtained for the samples, which illustrate great potential for practical application.