The invention discloses an effective method for extracting cucumis sativus mitochondria and DNA of the cucumis sativus mitochondria, and belongs to the field of plant biotechnology. Cucumis sativus etiolated seedlings are taken as materials, tissue homogenization buffer components and differential centrifugal force combination in the extraction process are adjusted, combined with percoll density gradient centrifugal separation, the mitochondria are obtained, 3% CTAB is used for pyrolyzing the mitochondria, and chloroform-isoamyl alcohol is subsequently used for extraction to obtain high-quality mtDNA. The mitochondria extracted by a homogenization buffer A prepared from mannitol and cysteine instead of saccharose and mercaptoethanol in a traditional method are good in integrity, obvious mitochondrion polymerization layers are obtained when centrifugal force for separating cell broken issue is 2000*g and centrifugal force for purifying the mitochondria is 17000*g, the concentration of the purified mtDNA is 158 ng*[mu]L<-1>, and 7.9 [mu]g of the mtDNA can be obtained through each 20 g of leaves. A260/A280 is about 1.87, and it is explained that the mtDNA extracted and separated through the method is high in purity. A set of effective system suitable for cucumis sativus mtDNA extraction and purification is established for the first time, and the effective method is of reference significance for establishing an mtDNA extraction and purification system for other species.