pUDK‐HGF, recombinant plasmid DNA encoding human HGF (hepatocyte growth factor), is a potential agent for gene therapy of ischaemic disease. Production of pUDK‐HGF is essential for its clinical application. In the present paper, a large‐scale manufacturing process was developed, including fermentation, cell harvest, alkaline lysis, capturing plasmid DNA with Q‐Sepharose XL chromatography, size‐exclusion chromatography on a Sephacryl S1000 column and refining with Source 15Q anion‐exchange chromatography. The quality criteria of pUDK‐HGF such as purity, concentration, homogeneity, residual RNA, chromosomal DNA, contaminated protein, endotoxin and HGF expression efficacy all were analysed and met the requirements for pharmaceutical‐grade plasmid DNA.