Human Esophageal squamous cell carcinoma (ESCC) represents a type of malignant tumor characterized by a high mortality rate and a generally poor surgical prognosis. The accurate labeling and high-specificity visualization of ESCC cells is extremely importance for its precise diagnosis and effective treatment. Fluorescence molecular imaging has emerged as one of the most critical modalities for cancer detection and therapeutic guidance, owing to its superior sensitivity, cost-effectiveness, portability, real-time imaging, and no damage. In this study, we initially verified that Hepsin, a protease, is highly expressed in ESCC through high-throughput immunocapture (HIC) and Western blot (WB) assays. Subsequently, we designed and synthesized an innovative activatable fluorescent probe, Ac-KQLR Rhodamine 110. It is specifically identified and cleaved by Hepsin, which is over-expressed in ESCC cells. Consequently, the Ac-KQLR Rhodamine 110 could be utilized for the activation fluorescence imaging of ESCC cells, providing a method for their precise visualization. In conclusion, this research highlights that the overexpression of Hepsin serves as a novel biomarker for ESCC. Based on Hepsin's high expression in ESCC, our team has developed a distinctive activation fluorescence imaging strategy that can be employed for the tracking and identification of ESCC. The implementation of this strategy could potentially revolutionize the current methodologies used for monitoring and treating cancer, thereby offering new hope and improved outcomes for patients diagnosed with ESCC.