Acetylcholinesterase is a well‐known enzyme, important in neurosciences and toxicology. Detection of AChE illustrates the complexity of quantifying proteins, even enzymes that have remarkable properties and substrates. AChE remains a model to propose novel strategies to detect proteins by light. Here we derived fluorescent probes from huprine, one of the most evolved AChE inhibitors. Two Near Infra‐Red (NIR) fluorescent probes HupNIR1 and HupNIR2 based on the huprine scaffold and cyanine 5.0 dyes have been synthesized and evaluated in situ for the detection of acetylcholinesterases in different tissues. As anticipated by the initial properties of huprine, both probes displayed a high affinity and selectivity for AChE vs BChE, with IC50 values in the nanomolar range and no non‐specific binding in the tissues. HupNIR2 appears the best probe for AChE with a great selectivity and sensitivity for AChE even in a low AChE region like striatum. Moreover, the binding of HupNIR2 is affected when AChE is inhibited with toxic molecules such as organophosphates. This work provided a new tool to visualize AChE activity in biological tissues.