Abstract: OBJECTIVE To investigate the trend of antibiotic resistance of extend-spectrum β-lactamases(ESBLs) producing Escherichia coli and Klebsiella pneumoniae in our hospital, and to provide a guidance for clinical rational use of antibiotics. METHODS Escherichia coli and Klebsiella pneumoniae strains were collected, which were isolated from patients in clinical in 2008-2009. Bacterial susceptibility testing were detected by MIC methods and ESBLs-producing strains were detected by double-disc diffusion confirming test. RESULTS In 2008, 312 of 764 strains of Escherichia coli were considered ESBLs, the rate of ESBLs were 40.8%; 133 of 356 strains of Klebsiella pneumoniae were considered ESBLs, the rate of ESBLs were 37.4%. In 2009, 246 of 499 strains of Escherichia coli were considered ESBLs, the rate of ESBLs were 49.3%; 107 of 298 strains of Klebsiella pneumoniae were considered ESBLs, the rate of ESBLs were 35.9%. The resistance rate of ESBLs- producing strains were significantly higher than that of ESBLs non-producing strains(P<0.05). The resistance rate of ESBLs-producing strains to aztreonam and cephalosporin were higher in 2009 than in 2008(P<0.05). The resistance rate of ESBLs-producing strains to quinolones were lower in 2009 than in 2008(P<0.05). The resistance rate of ESBLs-producing strains of Klebsiella pneumoniae to imipenem was increased by 15.9% in 2009(P<0.05). CONCLUSION ESBLs producing Escherichia coli and Klebsiella pneumoniae have the multidrug-resistant phenomenon, surveillance should be intensified, so as to avoid the increasing, diffusive and popular of ESBLs-producing bacteria.