Mesoporous alumina can serve as a framework for metal doping to form a novel nanocomposite. However, the nature of metal doping in amorphous mesoporous Al₂O₃ (aMA) is still obscured at the atomic level. This paper reports the one-pot synthesis of Cu-doped amorphous mesoporous alumina. Density functional theory calculations were carried out to reveal the geometric and electronic structure evolution upon Cu doping. Lattice distortion, metal distribution and charge compensation effects are the key factors responsible for the doping mechanism in aMA. The doped metal atoms prefer the predominant penta-coordinated (V) sites, and coexist with intrinsic defects, forming species with varied structures, influenced by the local structure, doping concentration and chemical environment. The most stable structures are formed under the balance of the above factors. The reduction of Cu⁺ to a metallic phase is hindered by the surrounding amorphous alumina reservoir. The dispersed Cu²⁺ species were proved to show higher catalytic activity.