The visualization and spatiotemporal monitoring of endogenous esterase activity are crucial for clinical diagnostics and treatment of liver diseases. Our research adopts a novel substrate hydrolysis-enzymatic activity (SHEA) approach using dicyanoisophorone-based fluorogenic ester substrates DCIP-R (R = R1−R6) to evaluate esterase preferences on diverse substrate libraries. Esterase-mediated hydrolysis yielded fluorescent DCIP−OH with a nanomolar detection limit in vitro. These probes effectively monitor ester hydrolysis kinetics with a turnover number of 4.73 s −1 and catalytic efficiency (k cat /K m ) of 10 6 M −1 s −1 (DCIP-R1). Comparative studies utilizing twophoton imaging have indicated that substrates containing alkyl groups (DCIP-R1) as recognition elements exhibit enhanced enzymatic cleavage compared to those containing phenyl substitution on alkyl chains (DCIP-R4). Time-dependent variations in endogenous esterase levels were tracked in healthy and liver tumor models, especially in diethylnitrosamine (DEN)−induced tumors and HepG2-transplanted liver tumors. Overall, fluorescence signal quantifications demonstrated the excellent proficiency of DCIP-R1 in detecting esterase activity both in vitro and in vivo, showing promising potential for biomedical applications.