A novel magnetic-responsive complex composed of polycation, DNA, and polyanion has been constructed via electrostatic interaction. The magnetic nanoparticles (MNPs) were first coated with a polycation, poly[2-(dimethylamino)ethyl methacrylate] end-capped with cholesterol moiety (Chol-PDMAEMA(30)), and then binded with DNA through electrostatic interaction; the complexes were further interacted with the brush-type polyanion, namely poly[poly(ethylene glycol)methyl ether methacrylate]-blockpoly[methacrylic acid carrying partial mercapto groups] (PPEGMA-b-PMAA(SH)). The resulting magnetic particle/DNA/polyion complexes could be stabilized by oxidizing the mercapto groups to form cross-linking shell with bridging disulfide (S-S) between PPEGMA-b-PMAA(SH) molecular chains. The interactions among DNA, Chol-PDMAEMA coated MNPs, and PPEGMA-b-PMAA(SH) were studied by agarose gel retardation assay. The complexes were fully characterized by means of zeta potential, transmission electron microscopy (TEM), dynamic light scattering (DLS) measurements, cytotoxicity assay, antinonspecific protein adsorption, and in vitro transfection tests. All these results indicate that this kind of magnetic-responsive complex has potential applications for gene vector.