Acetylcholinesterase (AChE) is a pivotal enzyme that is closely related with multiple neurological diseases, such as brain disorders or alterations in the neurotransmission and cancer. The development of convenient methods for imaging AChE activity in biological samples is very important to understand its mechanisms and functions in a living system. Herein, a fluorescent probe exhibiting emission in the near-infrared (NIR) region is developed to detect AChE and visualize biological AChE activities. This probe exhibits a quick response time, reasonable detection limit, and a large Stokes shift accompanied by the NIR emission. The probe has much better reactivity toward AChE than butyrylcholinesterase, which is one of the significant interfering substances. The outstanding specificity of the probe is proved by cellular imaging AChE activity and successful mapping in different regions of zebrafish. Such an effective probe can greatly contribute to ongoing efforts to design emission probes that have distinct properties to assay AChE in biological systems.