A new turn-on Cu²⁺ fluorescent sensor (CST) having a trehalose moiety, which confers a relatively large solubility in water, has been synthesized. The chemosensor is therefore suitable for studies in aqueous solution. Full potentiometric and UV-vis characterization evidence that at physiological pH CST forms with Cu²⁺ a species with a 1 : 1 stoichiometry allowing for a straightforward correlation between CST response and copper(II) concentration. The presence of the trehalose unit does not negatively affect the selectivity of CST for Cu²⁺ over a series of metal ions of interest as proven by fluorescence measurements. The novel chemosensor, tested in differentiated neuroblastoma SH-SY5Y cells, is able to detect Cu²⁺ in the extracellular region, as well as to track copper transfer processes upon cell stimulation induced by cellular depolarization.