• atHMOSs with amino groups were synthesized by a sol-gel method and a subsequent hydrothermal treatment procedure. • The prepared atHMOSs exhibited well-defined three-layered hollow structure and uniform mesopores. • The amino group in atHMOS gives it the ability to load large amounts of gold nanoparticles. Hollow mesoporous spheres with hierarchical structure and functional groups were considerably interest in separation, catalysis, and biomedicine. In this work, aminated three-layered hollow mesoporous organosilica spheres (atHMOSs) are synthesized for the first time by successively co-assembling multiple organosilanes with surfactant and a subsequent hydrothermal treatment procedure. Electron microscopy shows that the prepared atHMOSs have a well-defined three-layered hollow structure with a monodisperse diameter (440 nm) and a uniform mesopores (4.1 nm). Importantly, owing to the doped amino groups successfully, the atHMOSs can effectively coordinate with gold ions, and thus gold nanoparticles loaded in atHMOSs are further prepared after reducing the adsorbed gold ions.