Butyrylcholinesterase (BChE), one of the critical human cholinesterases, plays crucial roles in numerous physiological and pathological processes. Accordingly, it is a striking and at the same time challenging target for bioimaging studies. Herein, we developed the first ever example of a 1,2-dixoetane-based chemiluminescent probe (BCC) for monitoring BChE activity in native biological contexts such as living cells and animals. BCC was initially shown to exhibit a highly selective and sensitive turn-on response in its luminescence signal upon reacting with BChE in aqueous solutions. Later, BCC was utilized to image endogenous BChE activity in normal and cancer cell lines. It was also shown through inhibition experiments that BChE can detect fluctuations of BChE levels successfully. In vivo imaging ability of BCC was demonstrated in healthy and tumor-bearing mice models. BCC enabled us to visualize the BChE activity in different regions of the body. Furthermore, it was successfully employed to monitor tumors derived from neuroblastoma cells with a very high signal to noise ratio. Thus, BCC appears as a highly promising chemiluminescent probe, which can be used to further understand the contribution of BChE to regular cellular processes and the formation of diseased states.