TtgR, a transcriptional repressor from Pseudomonas putida, plays a key role in regulating multidrug resistance by controlling the expression of genes in response to various ligands. Despite its broad specificity, TtgR represents a promising candidate for the development of transcription factor (TF)-based biosensors. In this study, we utilized TtgR and its native promoter region (PttgABC) as genetic components to construct TF-based biosensors in Escherichia coli. By coupling TtgR and PttgABC with egfp, we developed a biosensor responsive to diverse flavonoids. To enhance the selectivity and specificity of the biosensor, we genetically engineered a TtgR-binding pocket. Engineered TtgR variants exhibited altered sensing profiles, enabling the development of biosensors with tailored ligand responses. Computational structural analysis and ligand docking provided insights into the interaction mechanisms between TtgR variants and flavonoids. Notably, biosensors based on wild-type TtgR and its N110F mutant were capable of quantifying resveratrol and quercetin at 0.01 mM with >90% accuracy. Although the precise molecular mechanisms involved remain unclear and further optimization is needed, the biosensors developed herein demonstrate strong potential for applications in numerous fields. This study lays the foundation for future research that could extend the utility of TtgR-based biosensors to synthetic biology, metabolic engineering, and beyond.