Oxidative stress in depression is a prime cause of neurotransmitter metabolism dysfunction in the brain. Acetylcholinesterase (AChE), a key hydrolase in the cholinergic system, directly determines the degradation of neurotransmitters. However, due to the complexity of the brain and lack of appropriate in situ imaging tools, the mechanism underlying the changes in AChE activity in depression remains unclear. Hence, we generated a two-photon fluorescence probe (MCYN) for real-time visualization of AChE with excellent sensitivity and selectivity. AChE can specifically recognize and cleave the carbamic acid ester bond in MCYN, and MCYN emits bright fluorescence at 560 nm by two-photon excitation at 800 nm. By utilizing MCYN to monitor AChE, we discovered a significant increase in AChE activity in the brains of mice with depression phenotypes. Notably, with the assistance of a two-photon fluorescence imaging probe of the superoxide anion radical (O-2(center dot-)), in vivo visualization for the first time revealed the positive correlation between AChE and O-2(center dot-) levels associated with depressive behaviors. This finding suggests that oxidative stress may induce AChE overactivation, leading to depression-related behaviors. This work provides a new and rewarding perspective to elucidate the role of oxidative stress regulating AChE in the pathology of depression.