Reduced nicotinamide adenine dinucleotide (NADH) and its phosphate ester (NADPH) are the factors involving in many redox reactions in living cells. In particular, the NAD(P)H plays a crucial role in the production of energy in the mitochondria by giving its electron. When NAD(P)H is defective, it causes degenerative diseases such as Parkinsonism and Alzheimer’s disease. In this regard, we developed a fluorescent probe 1 for imaging NAD(P)H in the mitochondrial of living cells. This probe consisted of red-emitting fluorophore as the signaling unit as well as NAD(P)H reactive site, and triphenyl phosphonium salt as mitochondria targeting group. Probe 1 is none-fluorescent, however, upon reacting with NAD(P)H a significant fluorescence increase was monitored in both solution and living human breast cancer cells (MDA-MB-231 cell). Furthermore, probe 1 can be used to differentiate between cancer (MDA-MB-231) and normal cells (NIH-3T3) based on mapping mitochondrial NAD(P)H-dependent fluorescence intensity. Probe 1 will be useful for various applications, ranging from elucidation of biological pathways to pathological diagnosis.