Mitochondria, as essential organelles in cells, play a crucial role in cellular growth and apoptosis. Monitoring mitochondria is of great importance, as mitochondrial dysfunction is often considered a hallmark event of cell apoptosis. Traditional fluorescence probes used for mitochondrial imaging and sensing are mostly intensity-based and are susceptible to factors such as concentration, the probe environment, and fluorescence intensity. Probes based on fluorescence resonance energy transfer (FRET) can effectively overcome external interference and achieve high-contrast imaging of mitochondria as well as quantitative monitoring of mitochondrial microenvironments. This review focuses on recent advances in the application of FRET-based probes for mitochondrial structure imaging and microenvironment sensing.