The invention relates to a purification method of Escherichia coli expressed CRM197 mutant, which belongs to the technology field of medicine. The purification method includes the following steps: performing denaturation and renaturation of inclusion body, ultra-filtering for concentrating, performing chromatography using anion exchange for purifying, and colleting protein peak to obtain the highpurity protein sample. The method is characterized in suitability for mass production, simple operation, short production period, high product purity, strong process stability, etc.