The ratio of oxidized to reduced NAD (NAD⁺/NADH) sets intracellular redox balance and antioxidant capacity. Intracellular NAD is compartmentalized and the mitochondrial NAD⁺/NADH ratio is intricately linked to cellular function. Here, we report the monitoring of the NAD⁺/NADH ratio in mitochondrial and cytosolic compartments in live cells by using a modified genetic biosensor (SoNar). The fluorescence signal of SoNar targeted to mitochondria (mt-SoNar) or cytosol (ct-SoNar) responded linearly to physiological NAD⁺/NADH ratios in situ. NAD⁺/NADH ratios in cytosol versus mitochondria responded rapidly, but differently, to acute metabolic perturbations, indicating distinct NAD pools. Subcellular NAD redox balance regained homeostasis via communications through malate-aspartate shuttle. Mitochondrial and cytosolic NAD⁺/NADH ratios are influenced by NAD⁺ precursor levels and are distinctly regulated under pathophysiological conditions. Compartment-targeted biosensors and real-time imaging allow assessment of subcellular NAD⁺/NADH redox signaling in live cells, enabling future mechanistic research of NAD redox in cell biology and disease development.