We have developed a two-photon fluorescent indicator (MC2P) that participates in a redox reaction with NADH to provide a fluorescence signal at 460 nm. The quantum yield of the indicator MC2P has increased about 26-fold in the presence of NADH. High two-photon efficiency (GM = 80.94) of the indicator MC2P has considered it as a unique device for assessing NADH expression in live cells without interference from native fluorogenic entities. It rapidly internalized into live cells and showed a considerable fluorescence signal in cancer cells than the immune cells (RAW 264.7). The indicator MC2P has provided information on the fluctuation of NADH levels depending on the variation of metabolites in the glycolysis pathway. Further, we noticed an increment of NADH level in mitochondrial respiratory cascade by inhibiting NADH-ubiquinone oxo reductase (complex I) using rotenone. On the contrary, uncoupler (CCCP) induced depletion of NADH. The dynamic expression of NAD(P)H has increased for counterbalancing mitochondrial redox potential during ROS (H2O2) suppression by GSH. Finally, in an vivo experiment, the indicator MC2P has distinguished colon tumor (HT-29 cells) tissue from healthy tissue by providing two-photon fluorescence images.