Abstract: Histone acetylation is an important epigenetic modification that governs gene expression, chromatin changes in stress response, and cell fate transition. FRET biosensors have been developed for various epigenetic events to enable spatiotemporal tracking of sub-cellular signaling events. Previously reported histone H3 acetylation biosensor recognizing two acetyl residues lacked specificity. In this study, using a single bromodomain of the BRD4, we have developed a genetically encoded H3K9ac biosensor. We systematically investigated different combinations of the BET family protein as binding domains and performed site-saturated mutagenesis to optimize the biosensor, achieving a dynamic FRET change up to 30% under TSA treatment. With the application of the optimized H3K9ac biosensor, we revealed different basal active chromatin architectures in invasive tumor cells compared to benign tumor cells. Furthermore, we found that H3K9ac level increased dramatically when cancer cells passed through microchannels, which models the physical constraints and mechanical microenvironmental conditions that cancer cells encounter when passing through narrow spaces within the body. This result highlights the chromatin plasticity in response to external mechanical stresses. In summary, our H3K9ac biosensor provides a versatile tool for mechanistic investigation of cell fate transition in cancer and mechanotransduction. Graphical Abstract