Carboxylesterase 2 (CES2) is a clinically significant serine hydrolase that governs the metabolic activation and detoxification of numerous ester-containing drugs and prodrugs. Despite its pivotal role in liver and colorectal cancer progression, direct in situ imaging of CES2 activity remains challenging due to the scarcity of probes with high isoform selectivity. To address this, we herein present XM-CE, a rationally engineered near-infrared (NIR) fluorescent probe specifically optimized for the highly sensitive and selective monitoring of CES2 activity. By integrating a CES2-responsive ester trigger with the XM-OH fluorophore, XM-CE yields a pronounced fluorescence turn-on response at 635 nm upon enzymatic hydrolysis. The probe demonstrates exceptional sensitivity with a limit of detection (LOD) of 0.023 U mL-1, alongside superior biocompatibility. These attributes enable high-resolution, real-time visualization of endogenous CES2 dynamics in living cells, zebrafish larvae, and tumor-bearing murine models. Collectively, our findings establish XM-CE as a robust analytical tool for mapping tissue-specific expression and monitoring enzyme regulation in vivo, offering significant potential for advancing biomedical research and clinical diagnostics.