Pathogenic bacterial infections pose a significant threat to human life, health, and socioeconomic development, with those arising from Escherichia coli (E. coli) O157:H7 being particularly concerning. Herein, customized aggregation-induced emission luminogens (AIEgen)-based signaling tags (TPA-galactose) were combined with a microfluidic chip for the determination of E. coli O157:H7. TPA-galactose undergoes hydrolysis by the β-galactosidase, resulting in the formation of highly fluorescent TPA–OH with AIE characteristics. Phages covalently bound to the surface of magnetic beads specifically capture and lyse E. coli O157:H7, releasing endogenous β-galactosidase, and the fluorescence intensity of TPA–OH facilitates the determination of E. coli O157:H7. The microfluidic chip process achieves a sensitivity of 45 CFU/mL in 45 min, requiring no DNA extraction or amplification, utilizing minimal sample volume, and enabling accurate one-stop quantification of live E. coli O157:H7. This strategy enables the rapid on-site determination of E. coli O157:H7 in environmental, food, and clinical samples, significantly enhancing public health and safety.