Abstract: A method for the isolated and sub-fractionation of the mitoehondria of Robinia pseudoacacia from plant tissue is described. The method comprised：（1） the blender tissue homogenization and the homogenate of tissue was obtained;（2） different centrifugation to remove impurities such as cell fragments;（3） add suspension, then, density gradient to obtain relatively pure mitochondria precipitation. Results showed that the integrity of mitochondrial was better by using this method and suitable for study on mitochondrial function as well as lay the foundation for the study of mitoehondrial proteomics.