Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), a crucial biomarker on the surface of tumor cells, plays a significant role in immune suppression and cancer metastasis. In this study, we successfully developed a novel amphiphilic fluorescent probe, QM-AMP, based on quinoline-malononitrile aggregation-induced emission (AIE) characteristics, for the real-time, and specific detection of ENPP1 enzymatic activity in multiple cell types. QM-AMP exhibits excellent dispersibility in both hydrophilic and hydrophobic environments, effectively minimizing background fluorescence interference and ensuring high sensitivity in ENPP1 detection. Upon interaction with ENPP1, the AMP group of the probe is specifically recognized and cleaved by ENPP1, which triggers the cleavage of the O–P bond and results in an “off-on” AIE fluorescence signal. QM-AMP demonstrates favorable photostability and biocompatibility, showing clear differential responses across cells with varying ENPP1 expression levels. Additionally, it can monitor ENPP1 expression changes under different macrophage polarization states, providing a potential tool for investigating ENPP1's role in the tumor immune microenvironment. This study not only offers a new approach for in-situ detection of ENPP1 as a biomarker but also opens up new possibilities for early diagnosis and treatment of related diseases.