A novel method involving ethanol-induced increase in the heterologous recombinant protein expression in E. coli cells was commonly used in recent studies. However, the detailed mechanism of this method is still to be revealed. This work used comparative transcriptomic analysis and numerous experiments to uncover the mechanism of ethanol effects on the expression of heterologous catalase in the recombinant strain E. coli BL21 (pET26b-katA). The key regulatory genes malK and prpD were found to have the most significant effects on the expression of heterologous catalase. Thus, the maltose ABC transporter and carbon metabolism from propanoate metabolism to citrate cycle were found to be the main regulatory pathways activated by ethanol to enhance the synthesis of heterologous proteins. Based on these mechanisms, a universally applicable E. coli expression host strain for improving the expression of heterologous proteins might be constructed.