A novel and eco-friendly procedure for synthesizing fluorescent carbon dots (CDs) functionalized with cuprizone and their application for the selective determination of Cu²⁺ are presented in this work. Water-soluble and fluorescent CDs were synthesized by a simple, low cost and green approach using sugarcane juice as a renewable natural source for the production of CDs. For the characterization of the CDs, UV-vis absorption spectroscopy, fluorescence spectroscopy, the dynamic light scattering technique, Fourier transform infrared spectroscopy, and transmission electron microscopy were used. The synthesized CDs have been functionalized with cuprizone (CPZ) in a simple, fast and single-step procedure. The functionalization eliminated all interference of the other studied metals and the CDs/CPZ was applied to the selective determination of Cu²⁺ in water. Under optimum conditions, a good linear relationship between the fluorescence intensity function and the concentration of Cu²⁺ in the range from 5.12 μM to 100 μmol L⁻¹ with a detection limit (S/N = 3) of 0.76 μmol L⁻¹ was obtained. Finally, this article proposes for the first time a factorial design to optimize the parameters for CD synthesis indicating that the luminescence intensity is a function of temperature and not of the heating time in the hydrothermal process.