Abstract: Considered the interference of biological background on fluorescence imaging in living cells, DCM-CBL, an esterase-activated near-infrared fluorescent prodrug, was designed and synthesized using dicyanopyran as the near-infrared fluorescent skeleton. The mechanism is as follows: esterase catalyzes the hydrolysis of the ester bond in probe structure, thereby separating the fluorophore from the anticancer drug, and at the same time activating the fluorescent signal at 695 nm, so that the detection of esterase and the monitoring of anticancer drug release can be achieved. Spectral experiment results show that DCM-CBL has a good fluorescence response to esterase in PBS buffer (pH 7.4, 10 mmol/L, containing 30% DMSO) and has excellent anti-interference and pH stability. Cell imaging confirmed that DCM-CBL can real-time monitor drug release in cancer cells, and also demonstrated excellent endoplasmic reticulum targeting (Pearson correlation coefficient is as high as 0.98); In addition, cytotoxicity experiments proved that DCM-CBL can selectively kill tumor cells to a certain extent.