Chymotrypsin, an extensively known proteolytic enzyme, plays a substantial role in maintaining physiological functions, including protein digestion, immune response, and tissue repair. To date, intense attention has been focused on the invention of efficient and sensitive chemical tools for chymotrypsin activity measurement. Among them, the "nonpeptide"-based chymotrypsin probe design strategy utilizing the esterase activity of chymotrypsin has been well-developed due to its low cost and high atom-economy feature. However, the ester-bond-based nature of these probes make them possibly vulnerable to esterases and active chemicals. These defects strictly restricted the application of the previously reported probes, especially for imaging in living systems. Therefore, to acquire fluorogenic probes with sufficient stability and specificity for chymotrypsin sensing in a complicated biological environment, a more stable skeleton for nonpeptide-based chymotrypsin probe construction is urgently needed. Herein, a novel nonpeptide-based fluorogenic probe for specific chymotrypsin activity sensing was designed and synthesized by the substitution of an ester-based linker with a heptafluorobutylamide moiety. The acquired probe, named TMBIHF, showed high selectivity toward various enzymes and reactive chemicals, while it retained high sensitivity and catalytic efficiency toward chymotrypsin. Moreover, TMBIHF was successfully applied for monitoring chymotrypsin activity and pancreas development in live zebrafish, specific sensing of exogenous and endogenous chymotrypsin in nude mice, and visualizing chymotrypsin-like activity-dependent cellular apoptosis, thus providing an alternative and reliable way for chymotrypsin-targeted biosensor or prodrug construction.