Using the fresh young leaves collected from cultivated strawberry (Fragaria × ananassa Duch. ‘Benihoppe’) as material, we separated and purified the chloroplasts by high saltlow pH buffer followed by Percoll density gradient centrifugation. The chloroplast DNA (cpDNA) was extracted by SDSProtein K method. The results of fluorescence microscope examination, agarose gel electrophoresis and PCR amplification showed that this method could obtain high integrity chloroplasts and high quality and purity cpDNA, which could meet the basic requirements of subsequent genome sequencing experiments. It could provide reference for extraction cpDNA of Fragaria and other herbaceous plants.