The present study reports the preparation of the first multivalent iminosugars built onto a glyco-gold nanoparticle core (glyco-AuNPs) capable of stabilizing or enhancing the activity of the lysosomal enzyme GCase, which is defective in Gaucher disease. An N-nonyltrihydroxypiperidine was selected as the bioactive iminosugar unit and further functionalized, via copper-catalyzed alkyne-azide cycloaddition, with a thiol-ending linker that allowed the conjugation to the gold core. These bioactive ligands were obtained with either a linear monomeric or dendritic trimeric arrangement of the iminosugar. The concentration of the bioactive iminosugar on the gold surface was modulated with different amounts of a glucoside bearing a short thiol-ending spacer as the inner ligand. The new mixed-ligand coated glyco-AuNPs were fully characterized, and those with the highest colloidal stability in aqueous medium were subjected to biological evaluation. Glyco-AuNPs with trimeric iminosugar bioactive units showed the ability to stabilize recombinant GCase in a thermal denaturation assay, while Glyco-AuNPs with monomeric iminosugar bioactive units were able to enhance the activity of mutant GCase in Gaucher patient's fibroblasts by 1.9-fold at 2.2 μM.