Neutrophil elastase (NE), a serine proteinase, is a significant biomarker which is closely related to the progress of diseases. However, only few probes have been reported for detection of NE activity and cell imaging. And these probes have exhibited small Stokes shift, which leads to high fluorescence interferences. Furthermore, only one probe among them is able to image NE in vivo successfully. To overcome the above problems, we designed a novel coumarin-based fluorescent probe HNCOU-NE with large Stokes shift to visualize NE activity in living cells and zebrafish. The new probe HNCOU-NE for NE contains fluorophore HNCOU as the reporter and pentafluoroethyl as the enzyme-active trigger moiety. As expected, HNCOU-NE displays perfect detecting performance for sensing of NE, including good water solubility, large Stokes shift, high affinity and wide linear response concentration. In addition, HNCOU-NE has been successfully utilized for NE real-time detection and imaging in different living cells, exhibiting low cytotoxicity and excellent biocompatibility. Most importantly, endogenous NE fluorescence imaging experiments reveals that HNCOU-NE can distinguish liver cancer cells (HepG2) and other cells (293T, HeLa and SKOV3), illustrating its specific ability to diagnose liver cancer cells. Besides, probe HNCOU-NE also has the ability to specifically detect endogenous NE activity in living zebrafish. All the results indicate that HNCOU-NE is a valuable probe for qualitative and quantitative sensing of NE activity in vitro and in vivo.