Objective Survey of hospital lactamase enzyme β-lactamase of Escherichia coli and Klebsiella pneumoniae clinical distribution and drug resistance. Methods Collected in our hospital from January 2007 to January 2010 clinical isolates of Escherichia coli and Klebsiella pneumoniae 401 and 319, respectively, by disk diffusion method (K-B method) of the isolates and susceptibility to antibiotics ESBLs screening and confirmatory test, and the clinical distribution and resistance of statistical analysis. Result 720 Escherichia coli and Klebsiella pneumoniae bacteria, the yield of extended spectrum β-lactamase-positive rate was 37.9%; which E. coli-positive rate was 42.1%; Klebsiella pneumoniae positive rate was 32.6 %.Producing extended spectrum β-lactamases in Escherichia coli and Klebsiella pneumoniae were the third generation cephalosporins, and monocyclic β-lactam antibiotics showed high resistance to aminoglycosides, quinolones, sulfonamides There are also high resistance to antibiotics, but susceptible to imipenem. Conclusion Court of Escherichia coli and Klebsiella pneumoniae the detection rate of ESBLs and showed multiple drug resistance, these strains produce ESBLs in different wards of the various samples, clinical should grasp their clinical distribution and drug resistance characteristics, rational use of antibiotics to control the spread and prevalence of ESBLs strains.