Overexpression of acetylcholinesterase (AChE) plays a pivotal role in disrupting acetylcholine (ACh) signaling, contributing to the neurodegenerative processes observed in Alzheimer's disease (AD). Detecting AChE activity is crucial for understanding disease progression and developing effective therapeutic strategies. In this study, we present a novel fluorescent probe, DPHP-AChE, which leverages large Stokes shifts (188 nm) and ratiometric fluorescence outputs based on aggregation-induced emission (AIE) and excited-state intramolecular proton transfer (ESIPT) mechanisms. This probe enables the efficient and reliable imaging of AChE activity in living systems, offering enhanced sensitivity and increased resistance to aggregation-caused quenching (ACQ). The large Stokes shift improves the signal-to-noise ratio and facilitates more accurate real-time quantification of AChE expression. We believe that this innovative approach opens up new possibilities for the specific detection of AChE, which can be further extended to the diagnosis and monitoring of AChE-related pathologies.