Abstracts Magnetic composite nanospheres carrying amino groups have attracted much interest due to their potential applications in the fields of catalysis, sensor, water purification, enzyme immobilization, DNA extraction, and drug delivery. In this paper, dedoped Fe3O4/polypyrrole (PPy) nanospheres with core-shell structure, high magnetization, superparamagnetism, and size meeting the demand of biorelated applications in vivo have been achieved by an in situ polymerization approach. The amount of the surface amino groups of Fe3O4/PPy is greatly enhanced by dedoping treatment due to liberation of the amino groups ever sheltered by the dopants, a situation that is responsible for facile conjugation of fluorescein and methotrexate (MTX) onto the magnetic composite through the EDC/NHS coupling chemistry to produce the desirable fluorescent Fe3O4/PPy/MTX targeting drug with a high MTX loading of ca. 109 μg mg-1. The as-obtained magnetic targeting drug exhibits controlled drug release, enhanced intracellular drug retention, and specific targeting ability to HeLa cells, presenting a promising candidate for treatment of cancer cells overexpressing folate receptors.