Abstract: Objective To develop and evaluate a novel deep sequencing method for mitochondrial DNA ( mtDNA) based on mitochondrial isolation and purification. Methods The optimal conditions for isolating mtDNA from cells were explored. Then, mtDNA from peripheral blood mononuclear cells ( PBMC) was deeply sequenced. The purity of mtDNA in the sequencing library was evaluated by the proportion of mtDNA reads in the sequencing fragments ( reads). The accuracy of mtDNA mutations detected by the established method was verified by Sanger sequencing. Results The mtDNA obtained by using the lysate containing low concentration of NP40 and without Tween-20 had high purity and almost no nuclear genome. The established method was used to detect six samples of PBMC, and the results showed that the proportion of mtDNA reads ranged from 87.8% to 92.8%, and that multiple mtDNA mutations were detected.One heterogenetic variation and three homogenetic variations were verified by Sanger sequencing, and the results were consistent. Conclusion The established deep sequencing method for mtDNA based on mitochondrial isolation and purification can be applied to PBMC samples and accurately and sensitively detect the heterogeneity and homogeneity of mitochondrial genome.