Naturally occurring glycoconjugates possess carbohydrate moieties that fulfill essential roles in many biological functions. Through conjugation of carbohydrates to therapeutics or imaging agents, naturally occurring glycoconjugates are mimicked and efficient targeting or increased cellular uptake of glycoconjugated macromolecules is achieved. In this work, linear and cyclic glucose moieties were functionalized with methacrylates via enzymatic synthesis and used as building blocks for intramolecular cross-linked single-chain glycopolymer nanoparticles (glyco-SCNPs). A set of water-soluble sub-10 nm-sized glyco-SCNPs was prepared by thiol-Michael addition cross-linking in water. Bioactivity of various glucose-conjugated glycopolymers and glyco-SCNPs was evaluated in binding studies with the glucose-specific lectin Concanavalin A and by comparing their cellular uptake efficiency in HeLa cells. Cytotoxicity studies did not reveal discernible cytotoxic effects, making these SCNPs promising candidates for ligand-based targeted imaging and drug delivery.