Cancer is still one of the important diseases that threatens the health of people. Multidrug resistance(MDR) is the main factor that leads to the failure of cancer chemotherapy. Thus, MDR diagnosis could facilitate the monitoring of the therapy process and realization of efficient treatment of tumors. In this study, we have tried to use a new tetrathiafulvalene(TTF) derivative(TTF-(COONBu4)2) to sensitively recognize the MDR through the multi-signal responsive strategy. The relevant electrochemical and spectroscopic studies demonstrate the specific binding behavior of TTF-(COONBu4)2 with P-glycoprotein(P-gp) as well as drug-resistant leukemia cells. Especially due to the over-expression of specific components of P-gp on the plasma membranes of drug resistant cells, the electrochemical and hydrophilic/hydrophobic features of drug resistant-leukemia cells are apparently different from those of other kinds of leukemia cells. Meanwhile, Fourier transform infrared spectroscopic study illustrates that the most intense vibration band of TTF moieties in the 1400–1600 cm-1 range is almost smeared out upon binding to P-gp, and the binding of TTF-(COONBu4)2 to P-gp may also lead to changes in protein secondary structure and backbone. This observation may advance the development of the new TTF agent for the promising clinical diagnosis and monitoring of MDR of tumors with the aim of successful chemotherapy for human cancer. Cancer is still one of the important factors that leads to the failure of cancer chemotherapy. Thus, MDR diagnosis could facilitate the monitoring of the therapy process and realization of efficient treatment of the tumors. In this study, we have tried to use a new tetrathiafulvalene (TTF) derivative (TTF- (COONBu4) 2) to sensitively recognize the MDR through the multi-signal responsive strategy. The relevant electrochemical and spectroscopic studies demonstrate the specific binding Behavior of TTF- (COONBu4) 2 with P-glycoprotein (P-gp) as well as drug-resistant leukemia cells. Especially due to the over-expression of specific components of P-gp on the plasma membranes of drug resistant cells, the electrochemical and hydrophilic / hydrophobic features of drug resistant-leukemia cells are apparently different from those of other kinds of leukemia cells. Meanwhile, Fourier transform infrared spectroscopy study illust rates that the most intense vibration band of TTF moieties in the 1400-1600 cm-1 range is almost smeared out upon binding to P-gp, and the binding of TTF- (COONBu4) 2 to P-gp may also lead to changes in This observation may advance the development of the new TTF agent for the promising clinical diagnosis and monitoring of MDR of tumors with the aim of successful chemotherapy for human cancer.