Here we report the template-free growth of hierarchical MnO₂ spheres on indium tin oxide by electrodeposition for the first time. The structural evolution and growth mechanism of MnO₂ was carefully investigated. The hierarchical MnO₂ showed mesoporous structure and good biocompatibility for the immobilization of glucose oxidase (GOx). The bioconjugate of a GOx/MnO₂-modified electrode was successfully employed for the mediatorless biosensing of glucose.