Esterases are common (biological catalyst) that play an essential role in regulating the physiological processes of organisms, in addition to contributing to disease prevention. Herein, we report the design and synthesis of an esterase-activated chemiluminescent probe (CL-EA) for the selective detection of esterases. Using the 1,2-dioxetane scaffold as the chemiluminescent platform and acetoxymethyl ether as the recognition group, the present probe was successfully lighted in the presence of esterases. As a result, a strong chemiluminescent signal was generated in an aqueous solution, thereby allowing the highly sensitive and specific detection of esterases. Furthermore, the probe is used to screen the activity of three plastic-degrading enzymes and image esterases in live cells and in a mouse model.