Carboxylesterase 2 (CES2), an endoplasmic reticulum (ER) located phase I enzyme, plays a vital role in the metabolism of various endogenous and exogenous substances, and is regarded as an important target for the design of prodrugs. Unfortunately, superior highly selective ER targeting fluorescent probes for monitoring of CES2 are not currently available. Herein, we report an ER targeting CES2 selective and sensitive ratiometric fluorescent probe ERNB based on the ER localizing group p-toluenesulfonamide. ERNB possessed high specificity, sensitivity, and exhibited excellent subcellular localization when compared to commercial ER tracker, and was used to image CES2 in the ER of living cells. Additionally, using ERNB we evaluated the CES2 regulation under d,l-dithiothreitol and tunicamycin-induced ER stress. Furthermore, we determined the down regulation of CES2 activity and expression in the acetaminophen-induced acute liver injury model. On the basis of these results, we conclude that ERNB is a promising tool for highlighting the role of CES2 in the ER and in exploring the role of CES2 in the development of diseases associated with ER stress.