This study reports an integrated microfluidic system capable of automatic extraction and analysis of mitochondrial DNA (mtDNA).....Alterations of mtDNA have been reported to be strongly associated with mitochondrial dysfunction, mitochondria-related diseases, aging, and many important human diseases such as diabetes and cancers. Thus, an effective tool for automatic detection of mtDNA deletion is in great need. This study, therefore, proposed a microfluidic system integrating three enabling modules to perform the entire protocol for the detection of mtDNA deletion. Crucial processes which included mtDNA extraction, nucleic acid amplification, separation and detection of the target genes were automatically performed. When compared with traditional assays, the developed microfluidic system consumed fewer samples and reagents, achieved a higher mtDNA extraction rate, and could automate all the processes within a shorter period of time (150 minutes). It may provide a powerful tool for the analysis of mitochondria mutations in the near future.