Carboxylesterase (CEs), as a key enzyme in ester metabolism, is simultaneously found with varying expression levels in diverse biological samples from a single organism, such as tissues, cells, bacteria and blood. However, the lack of integrated universal tools for the comprehensive detection of CEs'activity fluctuations in diverse biological samples from a single organism severely hinders the diagnosis and treatment of related diseases. Herein, we have developed an amphiphilic fluorescent probe (AZU-β) targeted toward CEs using an azulene derivative (AZU-OH) as a fluorophore. Using a "hydroxyl protection-deprotection" strategy, AZU-β incorporates a specific recognition group (acetyl ester) that activates the intramolecular charge transfer process to regulate the recognition signal toward CEs. AZU-β exhibits selectivity and highly sensitivity (the minimum detection limit is 1.8 × 10-2 U/mL), as well as rapid response (within approximately 6.0 s), for detecting CEs'activity over a wide range from 1.8 × 10-2 U/mL to 1.0 U/mL. Moreover, AZU-β exhibits outstanding water-oil amphiphilicity which makes it suitable for different biomembrane permeability levels. Therefore, AZU-β serves as an integrated universal tool that can not only detect CEs'activity at the serum level but also at cellular, tissue and bacterial levels under drug-induced liver injury conditions enabling the simultaneous monitoring of fluctuations in diverse biological samples from a single organism. It is expected that more probes targeting various disease-associated enzymes can be designed based on this amphiphilic design strategy to monitor relevant enzyme activity fluctuations in diverse biological samples from a single organism providing advanced analytical tools for related pathological research and diagnosis.