Magnesium(II) plays catalytic, structural, regulatory, and signaling roles in living organisms. Abnormal levels of this metal have been associated with numerous pathologies, including cardiovascular disease, diabetes, metabolic syndrome, immunodeficiency, cancer, and, most recently, liver pathologies affecting humans. The role of Mg2+ in the pathophysiology of liver disease, however, has been occluded by concomitant changes in concentration of interfering divalent cations, such as Ca2+, which complicates the interpretation of experiments conducted with existing molecular Mg2+ indicators. Herein, we introduce a new quinoline-based fluorescent sensor, MagZet1, that displays a shift in its excitation and emission wavelengths, affording ratiometric detection of cellular Mg2+ by both fluorescence microscopy and flow cytometry. The new sensor binds the target metal with a submillimolar dissociation constant─well suited for detection of changes in free Mg2+ in cells─and displays a 10-fold selectivity against Ca2+. Furthermore, the fluorescence ratio is insensitive to changes in pH in the physiological range, providing an overall superior performance over existing indicators. We provide insights into the metal selectivity profile of the new sensor based on computational modeling, and we apply it to shed light on a decrease in cytosolic free Mg2+ and altered expression of metal transporters in cellular models of drug-induced liver injury caused by acetaminophen overdose.