The invention relates to a large-scale method for extracting endotoxin-removed plasmids, which comprises firstly obtaining crudely separated plasmid DNA by classical alkaline lysis, and then adding isopropanol, NH 4 Ac solution, RNase A, NaCl, and then add the mixed solution of PEG6000 and NaCl, during the period, use TE solution to dissolve the precipitate, re-precipitate with absolute ethanol, wash the precipitate with 70% ethanol and cooperate with centrifugation, and finally dissolve the precipitate with TE solution after drying , to obtain the target plasmid. The invention can well remove the pollution of endotoxin to plasmid DNA. All the reagents used in the invention are chemical reagents that are always in the laboratory. The operation method is simple and easy to control, and is suitable for large-scale plasmid extraction. The concentration of the plasmid obtained by the present invention can reach 5mg / mL, and the endotoxin is lower than 5EU / μg, and the plasmid can be used for enzyme digestion, DNA sequence sequencing, cell transfection, virus packaging and clinical animal immune experiments.