ObjectiveTo study the resistance to commonly used antimicrobial agents and ampicillin resistance mechanism of Haemophilus influenzae (H. influenzae). MethodsAntimicrobial resistance and βlactamase of H. influenzae isolated from inpatients and outpatients in a hospital from January 1 to December 31, 2012 were detected by BauerKirby disc diffusion method and nitrocefinbased test, TEM and ROB βlactamase genes were amplified by polymerase chain reaction (PCR). ResultsOf 100 patients infected with H. influenzae, 61.00% were aged 1-10 years. The resistance rate to ampicillin was 35.00%(n=35)，resistance to sulphamethoxazole/trimethoprim was up to 64.65%；susceptibility rate to levofloxacin, azithromycin, ciprofloxacin, cefotaxime, chloramphenicol, cefuroxime, ampicillin / sulbactam   was 97.96%, 96.84%,96.91%, 92.78%,85.71%,77.89% and 74.75% respectively. Of 100 H.influenzae isolates, 21 were  βlactamase positive strains, and all were resistant to ampicillin. 35 ampicillinresistant strains were performed TEM and ROB genes detection, of which 22 were TEM positive, ROB positive strain was not found. ConclusionH.influenzae is sensitive to most antimicrobial agents except sulphamethoxazole/trimethoprim. Producing of TEM βlactamase is the major mechanism of ampcillin resistance in H.influenzae.