Hierarchical SiO₂–QDs@SiO₂ (QDs = CdTe, PbTe or PbS) nanostructures have been prepared by an integrated synthesis and self-assembly strategy using 3-mercaptopropionic acid (MPA) to stabilize the QDs and NaOH to control the self-assembly of QDs on the SiO₂ nanobead carriers. 3–10 nm QDs covered with a 1–2 nm SiO₂ shells were separately embedded on the surface of 50 nm SiO₂ nanobead carriers, resulting in hierarchical SiO₂–QDs@SiO₂ nanostructures. MPA and NaOH played important roles in the nucleation and growth of QDs, etching the SiO₂ nanobead carriers to the colloidal surface, and the self-assembly of the QDs on viscous SiO₂ nanobead carriers. The hierarchical SiO₂–QDs@SiO₂ nanostructures possess various advantages: (I) the growth of QDs and their self-assembly on SiO₂ nanobead carriers were performed in a one-pot reaction by a facile aqueous phase refluxing approach; (II) the SiO₂ shell with 1–2 nm in thickness can prevent the leakage of toxic Cd²⁺ or Pb²⁺ and avoid photobleaching or environmental pollution; (III) the surface of hierarchical SiO₂–QDs@SiO₂ nanostructures can be easily functionalized through silane-coupling chemistry. Because of the excellent photoluminescence and biocompatibility, the hierarchical SiO₂–QDs@SiO₂ nanostructures modified by carboxyl (–COOH) active group can be available for covalent coupling to various biomolecules, which have shown potential applications in fingerprint detection and in vivo cell imaging.