In the current work, a near-infrared (NIR) fluorescent probe (CyClCP) was developed for fast (35 min), highly
sensitive (LOD of 3.75 U/L) and selective response to BChE in vitro and in vivo. Upon the addition of BChE,
CyClCP could be efficiently activated with remarkable NIR (λem= 708 nm) fluorescence enhancement and
obvious absorbance red shift (581 nm–687 nm). Specifically, according to the subtle differences structural
features and substrate preference between BChE and its sister enzyme AChE, CyClCP was constructed by in-
troducing chlorine atom at the ortho-position of the phenolic hydroxyl in the previous reported probe (CyCP).
Fortunately, CyClCP exhibited better selectivity towards BChE over AChE compared with CyCP. This molecular
design strategy was further rationalized by docking molecular of fluorescence probes (CyClCP and CyCP) and
enzymes (BChE and AChE). Finally, CyClCP was membrane permeable and successfully applied to image en-
dogenous BChE level in HepG2 and LO2 cells. Therefore, CyClCP could serve as a promising tool for BChE-
related physiological function studies in complex biological systems.