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Pathogenic <i>E. coli</i> infection is one of the most widespread foodborne diseases, so the development of sensitive, reliable and easy operating detection tests is a key issue for food safety. Identifying bacteria with a fluorescent medium is more sensitive and faster than using chromogenic media. This study designed and synthesized a <i>β</i>-galactosidase-activatable fluorescent probe BOD-Gal for the sensitive detection of <i>E. coli</i>. It employed a biocompatible and photostable 4,4-difluoro-3a,4a-diaza-s-indancene (BODIPY) as the fluorophore to form a <i>β</i>-<i>O</i>-glycosidic bond with galactose, allowing the BOD-Gal to show significant on-off fluorescent signals for in vitro and in vivo bacterial detection. This work shows the potential for the use of a BODIPY based enzyme substrate for pathogen detection.