KillerRed is a unique red fluorescent protein exhibiting excellent phototoxic properties. It has the ability to produce reactive oxygen species (ROS), for killing tumor cells in vitro upon laser irradiation and has the potential to act as a photosensitizer in the application of tumor therapy. Here, we investigated the effects of KillerRed-based photodynamic therapy (PDT) on tumor growth in vivo and examined the subsequent tumor metabolic states including the changes of pyridine nucleotide (PN) and flavoprotein (Fp), two important metabolic coenzymes of tumor cells. Results showed that the tumor was scabbed in response to 561 nm laser irradiation at 80 mV for 3 min, and the tumor growth had been significantly inhibited by KillerRed-based PDT treatment compared to control groups. More importantly, a home-made cryo-imaging redox scanner was used to measure intrinsic fluorescence and exogenous KillerRed fluorescence signals in tumors. The flavoprotein was remarkable elevated and the PN was seldom increased with concomitant photobleaching of KillerRed fluorescence after irradiation, suggesting that flavoprotein and PN were oxidized in the course of KillerRed-based PDT.