The invention discloses a detection method for mitochondrial copy number and sequence variation. Based on a BGI-Seq series sequencing platform, a high-flux, low-cost and automated mitochondrial variation detection technology is established, and the technology comprises mitochondrial whole-genome DNA separation and genome housekeeping gene amplification, library construction and sequencing, and supporting information analysis algorithm development. The technology can simultaneously achieve mitochondrial copy number variation and sequence variation detection, and is high in flux, small in difference between batches, high in accuracy, low in cost, short in used time, and capable of fully meeting the need of automated production. The technology can be applied to mtDNA somaclonal variation research and detection, diagnosis of mitochondrion related diseases, research of the role, played in the aging process, of mitochondria, tumor cell metabolic activity related research and the like, and has good prospects.