Abstract: Diaphorase is a protease commonly found in animal tissues that protects cells from damage by preventing production of superoxide anions induced by various free radicals. Because diaphorase is overexpressed in many tumor tissues, it becomes an important biomarker for early diagnosis and treatment of tumors. Using the characteristics of high specificity, high sensitivity and real-time in-situ detection of fluorescence sensing technology, we designed and synthesized a near-infrared fluorescence probe ( MITZ 1 ) based on trimethylquinone. In the presence of NADH, the probe reacted specifically with diaphorase to release fluorophore. The fluorescence value of the solution increased nearly 5 times at 770 nm, and the change of diaphorase content in the system was detected by obvious fluorescence signal response. MITZ 1 has the characteristics of high selectivity, high photostability, low toxicity and low detection limit, and has been successfully applied to the biological imaging detection of diaphorase in tumor cells (HeLa) and c. elegans.