The invention discloses a method for extracting mitochondrial deoxyribonucleic acid (
DNA) of cotton. The method comprises the following steps of: (1) homogenizing cotton, and centrifuging and collecting precipitate; (2) removing
nuclear DNA, adding
ethylene diamine tetraacetic acid (EDTA) solution, and centrifuging to obtain the precipitate; (3) adding
buffer solution, and centrifuging to obtain mitochondria; (4) adding
lysis solution and acetate solution, centrifuging, collecting supernatant, and extracting by using
chloroform-isoamylol; (5) adding acetate solution and absolute
ethanol, centrifuging to obtain the precipitate, and washing by using
ethanol; (6) dissolving in triisopropylphenylsulfonyl (
Tris)-EDTA (TE); (7) removing ribonucleic acid (
RNA) and
protein, extracting by using
Tris saturated
phenol-
chloroform-isoamylol, and centrifuging and collecting the supernatant; and (8) adding the acetate solution and the
ethanol, and centrifuging to obtain the precipitate; and washing by using the ethanol to obtain the mitochondrial
DNA. Aiming at defects of the conventional extraction of the mitochondrial
DNA, the conventional cetyltrimethylammonium
bromide (CTAB) method is improved, and the extraction conditions are optimized by adjusting the time and times of
centrifugation, changing reagents, increasing extraction and washing times, and the like. On the basis of ensuring that the mechanical damage degree of the mitochondria is minimum, the
nuclear DNA,
phenols and polysaccharides outside the mitochondria are effectively removed,
pollution rate is reduced, the purity and the quality of the mitochondrial DNA are improved, and the mitochondrial DNA can meet the requirement of subsequent experiments of the
gene engineering of the cotton mitochondria.