We describe a protocol to prepare a multiplexed mtDNA library from a blood sample for performing a long read sequencing of the mitochondrial genome. All steps are carefully described to get a high enrichment of mtDNA relative to total DNA extracted from the blood sample. The obtained mutiplexed library allows the production of long sequence mtDNA reads up to 16.5 kbp with a quality enabling variant-calling by using a portable sequencer (MinION, Oxford Nanopore Technologies).