Copper is an essential trace element for normal development and function throughout the body, including the central nervous system (CNS). Alterations to cellular copper levels result in severe neurological consequences and are linked to a range of CNS disorders, positioning treatments that restore copper balance as promising therapies for these disorders. However, despite the clear relationship between copper balance and CNS health, there are limited tools to measure copper levels in vivo in humans. This constitutes a significant challenge for both diagnosing disorders of copper imbalance and monitoring the efficacy of copper-altering treatments for these disorders. Here we report the synthesis and characterization of Fluorine-labeled Naphthalimide Copper sensor 1 (F-NpCu1), a fluorescent sensor for copper that contains a fluorine atom for future radiolabeling for clinical application. We demonstrate that the probe exhibits good stability and is highly selective for copper above other transition metals present in biological tissues. Copper binding promotes covalent bond formation between the sensor and proximal cellular proteins. F-NpCu1 is nontoxic and can be measured using fluorescence microscopy in living cells and fixed tissue sections from both mouse brain and pancreas. Furthermore, F-NpCu1 exhibits good blood-brain-barrier permeability and can report differences in brain copper levels induced by copper modulating therapies in living mice using intravital fluorescence microscopy. This study represents a promising advance toward the development of the first clinical tool for measuring copper in living humans, including in the CNS, with radiolabeling studies underway to develop 18F-NpCu1 for PET imaging of copper in vivo.