The present invention discloses a novel mitochondrial genome editing tool, and belongs to the field of genome engineering. According to the prevent invention, the constructed mtCRISPR/Cas9 system mainly comprises two parts such as gRNA entering mitochondria and Cas9 nuclease localized in mitochondria, wherein the constructed mt-gRNA has two forms, the one mt-gRNA comprises a RNA mitochondrial localizing guide sequence, a targeting sequence and a gRNA skeleton sequence, the other mt-gRNA comprises a RNA mitochondrial localizing guide sequence, any one tRNA sequence encoded by mitochondrial or other additional spacer sequences, a targeting sequence and a gRNA skeleton sequence, the obtained combined material acts on the mitochondrial genome to break the target sequence in a targeted manner after the mt-gRNA and the mtCas9 nuclease are combined, and the action efficiency of the second mt-gRNA action is high than the action efficiency of the mt-gRNA; and the results verify that the constructed mtCRISPR/Cas9 system has characteristics of high efficiency and strong specificity.