Abstract: Bacterial drug resistance has become a hot issue affecting the global public health in the 21st century, which is seriously affecting the treatment effect and the development of agriculture and animal husbandry. Since the rate of developing new antibiotics is much slower than the evolutional speed of drug-resistant bacteria, improving the bactericidal effect of traditional antibiotics is an important way to solve the complex problem of bacterial resistance. The killing test against Escherichia coli BW25113 was carried out by adding the exogenous alcohols and tobramycin. The results showed that the 1,2-butanediol obtained from the screening could significantly enhance the killing effect of aminoglycoside antibiotics against BW25113 and the clinical drug-resistant Escherichia coli. The tobramycin uptake experiments have confirmed that 1,2-butanediol could promote the uptake of tobramycin into the cell by Escherichia coli, cause the increase of intracellular antibiotics and then lead to the bacterial death, suggesting that its auxiliary sterilization effect could be achieved.