Recent advancements in biosensor technology have revolutionized the field of microbial engineering, enabling efficient and precise optimization of strains for the production of valuable chemicals. This review comprehensively explores the innovative integration of biosensors to enhance microbial cell factories, with a particular emphasis on the crucial role of high-throughput biosensor-assisted screening. Biosensor-assisted approaches have enabled the identification of novel transporters, the elucidation of underlying transport mechanisms, and the fine-tuning of metabolic pathways for enhanced production. Furthermore, this review illustrates the utilization of biosensors for manipulating cellular behaviors, including interactions with environmental factors, and the reduction of nongenetic cell-to-cell variations. This review highlights the indispensable role of biosensors in advancing the field of microbial engineering through the modulation and exploitation of diverse cellular physiological processes.