ABSTRACT Bacteria can adapt their lifestyles, including microbial growth, metabolism, and biofilm formation, in response to light signaling. However, the molecular pathways through which blue light affects the lifestyle of Escherichia coli ( E. coli ) remain incomplete and poorly understood. To address this gap, transcriptomic and proteomic approaches were employed to analyze the physiological differences of E. coli under dark and blue light conditions. Our results indicate that, compared to dark conditions, blue light attenuates flagellar assembly, reduces cell motility and communication, and decreases biofilm formation in E. coli . In addition, this study elucidates the signaling pathways involved in the blue light‐mediated regulation of E. coli behavior, providing a theoretical framework for understanding how E. coli responds to blue light signaling to modulate biofilm formation for the production of food chemicals.