Multivalent protein–glycan interactions are widespread in biology and are vital for initial recognition in cell–cell communication, host immune regulation, and viral and bacterial infection. Recently, fluorescent nanomaterials such as quantum dots (QDs) have been identified as a capable scaffold for multivalent carbohydrate immobilization. Carbon dots (CDs) are essentially heavy-metal-free QDs, offering a low-toxicity alternative for bio-imaging and biosensing applications. Herein, we report a simple and versatile route for linker functionalization of lactose with (3-glycidyloxypropyl)trimethoxysilane (GOPTS) for conjugation to various CDs. CDs derived by thermal treatment of polyethylenimine (PEI) with citric acid and lactose, as the glycan, were employed in a number of detailed biological applications and evaluations. The self-assembled glycan monolayer (SAGM) method described here resulted in a high yield of lactose-conjugated CDs. Specific interactions of our lactose-coated CDs with cells, lectin microarrays, and as internalized bioimaging nanolights were demonstrated, with intracellular localization in a variety of cell lines observed. This present study offers a facile, one-pot, green, and low-cost synthesis of fluorescent multivalent nanoparticles that can be applied in the study of diverse interactions across the glyco-interactome.