Abstract: In this study, yellowing Glycine max(L.) Merrill seedlings were used to extract mitochondrial DNA(mtDNA) by optimizing centrifugal force and time during differential centrifugation. Furthermore, establishing different sucrose density gradients and lysate concentrations during purification, combined with high salt removal protein, improved the extraction method of soybean mtDNA. Results showed that the concentration and purity of mtDNA extracted by this method were high, with no chloroplast or nuclear genomic DNA contamination, the mtDNA could be used for subsequent research on soybean mitochondrial genome.