Abstract: Studies on plasmid extraction of lactic acid bacteria have a long history. Up to date, a number of effective plasmid extraction methods of lactic acid bacteria have been reported. Most of them focused on Lactococcus, however, information on plasmid preparation technique of Lactobacillus plantarum is scare. In this study, the extracting efficiency of different plasmid preparation methods in Lactobacillus plantarum were comparatively investigated, and the reported plasmid extraction methods were optimized. A high-efficient native plasmid preparation method for Lactobacillus plantarum in plateau phase was developed. In addition, the number of native plasmid and high-copy-number plasmid of Lactobacillus plantarum were assessed, which provided technical references for the preparation of donor plasmid and the selection of receptor required in further construction of Lactobacillus plantarum expression system.