Objective To analyze the susceptibility of carbapenem-resistant Enterobacteriaceae (CRE) to polymyxin B by two different methods, so as to provide basis for the selection of antimicrobial susceptibility methods in laboratory and rational clinical amtimicrobial use. Methods 230 strains of CRE isolated from the First Affiliated Hospital of Kunming Medical University between January and December 2018 were collected, the susceptibility of CRE to polymyxin B was detected by E-test and broth microdilution(BMD) method respectively, differences between two methods were analyzed. Results The main source of bacteria was sputum (163 strains, 70.87%), followed by blood (33 strains, 14.35%) and secretion (15 strains, 6.52%), Klebsiella pneumoniae was the main pathogen (205 strains). Susceptibility rate and resistant rate of CRE strains detected by E-test were 96.52% and 3.48% respectively; susceptibility rate and resistant rate of CRE strains detected by BMD method were 95.65% and 4.35% respectively; the basic consistent rate of two methods was 95.65%, classification consistent rate was 99.13%, and very major error was 0.87%. Conclusion Polymyxin B has good antimicrobial activity against CRE, E-test method is easy to be operated and is well consistent with BMD method, it can be used in clinical routine antimicrobial susceptibility testing.