Human serum albumin (HSA) exhibits diverse enzymatic properties, with its esterase-like activity (HSAEA) playing a pivotal role in lipid metabolism and drug development processes. Herein, we developed a novel fluorescent probe, THOA, for the specific evaluation of HSAEA and albumin. Upon cleavage by HSAEA, THOA undergoes hydrolysis to yield THOH, which demonstrates heightened fluorescence intensity through a synergistic mechanism involving ESIPT and ICT. The THOA probe demonstrated outstanding selectivity, precise triple-linear response, and reliable performance in the quantification of HSAEA. When applied to serum samples from both healthy volunteers and nephrology patients, THOA showed a unique capability to identify subclinical cases characterized by normal binding capacity but aberrant HSAEA activity. Additionally, THOA was successfully employed for in-situ imaging of reabsorbed HSA in cells and zebrafish. This study provides crucial insights into assessing the functional status of HSA.
