Mitochondrial diseases are a group of highly heterogeneous genetic disorders that affect any organ at any age, caused by mutations in nuclear and mitochondrial genes, characterized by complex clinical manifestations and diagnostic challenges. Multi-omics technologies have emerged as essential tools for deciphering the molecular basis of these diseases and improving clinical outcomes. This review summarizes the research progress of multi-omics approaches—including genomics, transcriptomics, proteomics, metabolomics, and single-cell sequencing—in resolving genetic and phenotypic heterogeneity and elucidating cell- and tissue-specific mechanisms of representative mitochondrial diseases. We aim to provide a reference and framework for the translation of multi-omics technologies from basic research to clinical application within the field of mitochondrial medicine.