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Elevated butyrylcholinesterase (BChE) levels are recognized as an indicator of type 2 diabetes mellitus (T2DM), a finding supported by clinical trials. Technologies that utilize BChE as a biomarker for diagnosing T2DM are currently under development, yet few fluorescent probes have been explored in this field. Fluorescence imaging offers several advantages including ease of operation, rapid detection speed, and high sensitivity. Consequently, the development of a fluorescent probe capable of detecting BChE activity for T2DM holds significant importance. However, there is a lack of a specific fluorescent probe to detect the BChE level across multi-models, including cellular systems, zebrafish, and mice with T2DM. Herein, we designed a fluorescent probe that is specifically responsive to BChE, enabling effective detection of changes in BChE levels. The probe demonstrated outstanding sensitivity, specificity, and photostability, with its fluorescence intensity remaining largely unaffected by pH values. The probe effectively detected changes in cellular BChE levels induced by various inducers and successfully identified variations in the abdominal fluorescence signal in zebrafish treated with these inducers. Furthermore, the probe was employed to assess BChE levels in the tissues of the T2DM mouse model. The probe facilitates a deeper understanding of the pathogenesis and progression of T2DM and provides a powerful diagnostic tool for potential therapeutic targets.