Significance: Beyond their roles as redox currency in living organisms, pyridine dinucleotides (NAD⁺/NADH and NADP⁺/NADPH) are also precursors or cosubstrates of great significance in various physiologic and pathologic processes.

Recent Advances: For many years, it was challenging to develop methodologies for monitoring pyridine dinucleotides in situ or in vivo. Recent advances in fluorescent protein-based sensors provide a rapid, sensitive, specific, and real-time readout of pyridine dinucleotide dynamics in single cells or in vivo, thereby opening a new era of pyridine dinucleotide bioimaging. In this article, we summarize the developments in genetically encoded fluorescent sensors for NAD⁺/NADH and NADP⁺/NADPH redox states, as well as their applications in life sciences and drug discovery. The strengths and weaknesses of individual sensors are also discussed.

Critical Issues: These sensors have the advantages of being specific and organelle targetable, enabling real-time monitoring and subcellular-level quantification of targeted molecules in living cells and in vivo.

Future Directions: NAD⁺/NADH and NADP⁺/NADPH have distinct functions in metabolic and redox regulation, and thus, a comprehensive evaluation of metabolic and redox states must be multiplexed with a combination of various metabolite sensors in a single cell. Antioxid. Redox Signal. 28, 213–229.