Abstract: In order to screen the new drug-resistant key genes and study their drug resistance mechanisms to alleviate the problem of bacterial resistance, by constructing the overexpression strains of the ethanolamine-utilizing chaperone protein EutJ and the alcohol dehydrogenase EutG, the killing tests of gentamicin were performed on the two strains. Then, by taking the killing effect of the wild-type Escherichia coli carrying the empty plasmid in logarithmic phase as the control group, the minimum inhibitory concentration (MIC) was further determined, and the correlation between the ethanolamine metabolism and bacterial tolerance was studied through the detection of antibiotic uptake by inhibition zone. The results showed that after the overexpression of EutJ and EutG, Escherichia coli could reduce the concentrations of antibiotics in the bacteria to improve the survival rate under the condition of gentamicin killing, which proved that the genes of eutJ and eutG could be used as the potential tolerance-related genes to help the bacteria escape from the antibiotic attacks. Therefore, the key enzymes of ethanolamine metabolism, EutJ and EutG, were expected to be the targets for the development of immune adjuvants or new antibiotics.