Human carboxylesterase 2A (hCES2A) is an important endoplasmic reticulum (ER)-resident enzyme that is responsible for the hydrolytic metabolism or activation of numerous ester-bearing drugs and environmental toxins. The previously reported hCES2A fluorogenic substrates suffer from limited emission wavelength, low specificity, and poor localization accuracy, thereby greatly limiting the in situ functional imaging of hCES2A and drug discovery. Herein, a rational ligand design strategy was adopted to construct a highly specific near-infrared (NIR) substrate for hCES2A. Following scaffold screening and recognition group optimization, HTCF was identified as a desirable NIR fluorophore with excellent photophysical properties and high ER accumulation ability, while several HTCF esters held a high potential to be good hCES2A substrates. Further investigations revealed that TP-HTCF (the tert-pentyl ester of HTCF) was an ideal substrate with ultrahigh sensitivity, excellent specificity, and a substantial signal-to-noise ratio. Upon the addition of hCES2A, TP-HTCF could be rapidly hydrolyzed to release HTCF, a chemically stable product that emitted bright fluorescent signals at around 670 nm. A TP-HTCF-based biochemical assay was then established for the high-throughput screening of potent and cell-active hCES2A inhibitors from an in-house compound library. Furthermore, TP-HTCF displayed high imaging resolution for imaging hCES2A in living cells as well as mouse liver slices and tumor-xenograft mice. Collectively, this study demonstrates a rational strategy for developing highly specific fluorogenic substrates for an ER-resident target enzyme, while TP-HTCF can act as a practical tool for sensing hCES2A in living systems.