Nanosized gold nanoparticles (AuNPs) are of great interest in areas such as catalysts or imaging but are easy to aggregate due to high surface activity. To stabilize AuNPs, two approaches were employed to immobilize AuNPs in spherical polymer brushes (SPBs), namely, the in situ preparation of AuNPs within the brush layer of SPBs and external addition of preprepared citrate-capped AuNPs. The distribution and stability of AuNPs in SPBs were studied by small-angle X-ray scattering (SAXS). SAXS results demonstrated that the in situ-prepared AuNPs were mainly located on the inner layer and their amount decreased from inside to outside. In the case of external addition of preprepared AuNPs, the cationic SPB showed obvious immobilization, while almost no AuNPs were immobilized in the anionic SPB. The stable immobilization of the AuNPs in SPBs was the result of multiple interactions including complexation and electrostatic interaction. SAXS was validated to be a distinctive and powerful characterization method to provide theoretical guidance for the stable immobilization of AuNPs.