Abstract: The disclosure discloses a method for increasing the intracellular heme content of Escherichia coli and belongs to the field of metabolic engineering. In the disclosure, in E. coli, the gene mscS encoding a small conductance mechanically sensitive ion channel protein is knocked out, the gene aroG encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase is knocked out, or the gene hemA encoding glutamyl-tRNA reductase is overexpressed. The constructed recombinant strain is cultured in an LB culture medium, and the heme content can reach 47.6 ?mol·L?1, which is significantly higher than that of a control strain. The recombinant strain has the value of wide application.