The invention provides a method for measuring content of linear DNA of a nucleic acid medicine. The method comprises the steps of performing liquid chromatographic analysis on a to-be-detected nucleicacid medicine, and determining, based on a result of the liquid chromatographic analysis, the content of the linear DNA of the to-be-detected nucleic acid medicine. The method for measuring content of linear DNA of the nucleic acid medicine can separate and quantitatively analyze linear plasmid DNA in the nucleic acid medicine quickly, accurately and flexibly, can be used as a common quality control manner for nucleic acid medicines (sample in middle process, original liquid, semi-finished product and finished product), provides reliable quality control basis for production of the nucleic acid medicines, and highly supports research and development and safe production of recombined biological products, so as to ensure security of clinical medication.