Mitochondria play essential roles in cellular energy metabolism and biological processes, with their dysfunction implicated in various diseases. Mitochondria-targeted fluorescent probes have thus been developed to sensitively detect metabolites and monitor mitochondrial metabolic status in real time, aiding in the diagnosis and treatment of mitochondria-related diseases. To overcome limitations such as poor water solubility and instability of conventional probes, structurally optimized molecular fluorescent probes have been designed. In parallel, nanotechnology has facilitated the development of novel mitochondria-targeted nanofluorescence probes. Moreover, computer simulation and artificial intelligence have significantly accelerated probe design and optimization. This review summarizes recent advances in mitochondria-targeted fluorescence probes, emphasizing their structural design, functional properties, and molecular mechanisms. It also highlights the role of computational approaches in promoting innovative development in this field.