Biofilms enable bacterial cells to adhere and thrive on surfaces, with associated changes in growth and gene expression aiding their survival in challenging environments. While previous research has explored E. coli biofilm formation, there has been limited exploration of its application in industrial production. Prior studies have shown that immobilized fermentation can enhance L-threonine production. This study aims to augment biofilm formation and subsequently increase L-threonine production in E. coli by regulating the quorum sensing system, focusing on key AI-2-related genes, including luxS, lsrB, lsrK, and lsrR. In +pluxS and +plsrB strains, AI-2 levels were significantly altered, resulting in enhanced biofilm formation, increased curli expression, shorter free-cell fermentation periods, and improved production efficiency through immobilized continuous fermentation. In a single batch of free-cell fermentation with E. coli W1688, L-threonine production was 10.16 g/L. However, +pluxS and +plsrB strains achieved L-threonine yields of 15.27 g/L and 13.38 g/L, respectively, after seven fermentation batches. Additionally, the fermentation period was reduced from 36 h to 28 h and 30 h, respectively.