The abuse and misuse of antibiotics in medical treatment and livestock production have led to the generation of bacterial resistance, especially Escherichia coliresistance to ampicillin, which has compromised clinical treatment. To further understand the specific characteristics of E. coli resistance to ampicillin, we explored the growth and resistance characteristics of bacteria evolving from sensitive to resistant bacteria, with the aim of discovering new phenomena and patterns. In the present study, ampicillin-resistant E. coli strains with minimal inhibitory concentration (MIC) values of 4-, 16-, and 64-fold were obtained through artificial passage. Results showed that the diameter of the colony inhibition circle decreased at the same concentration of ampicillin and "microcolonization"appeared as bacterial resistance increased. Regarding growth rate, ampicillin-resistant bacteria grew more slowly than susceptible bacteria. The MIC value of ampicillin-resistant bacteria for cefpirome and cefazolin, which are both β-lactam antibiotics, increased as drug resistance increased. In addition, the intracellular concentration of ampicillin and the permeability of the cell membrane decreased as drug resistance increased. This study explored the changing drug resistance characteristics of E. coliampicillin-resistant bacteria to varying degrees, and provides a foundation for future studies investigating the resistance mechanism and clinical treatment of E. coli ampicillin-resistant bacteria.