Abstract: OBJECTIVE To investigate antimicrobial resistance and resistant mechanism of Haemophilus influenza to ampicillin in children, so as to provide evidence for clinical treatment. METHODS A total of 375 strains of H. influenza isolated from children with respiratory infections from Shanghai Children's Hospital from 2012 to 2013 were collected. Antimicrobial resistance and β-lactamase of H.influenzae were detected by Kirby-Bauer disk diffusion method and nitrocefin-based test. The β-lactamase genes were amplified by polymerase chain reaction (PCR). RESULTS The antimicrobial resistant rate of H. influenzae to ampicillin was 40.3%, and the susceptibility rates to amoxicillin/clavulanic acid, ampicillin/sulbactam, cefuroxime and cefotaxime were all higher than 70.0%. Of 375 isolates, 119 strains were β-lactamase positive, with the production rate of 31.7%, and TEM-1 gene was detected in ampicillin-resistant strains, with no ROB-1 gene positive being found. CONCLUSION The antimicrobial resistance of H. influenzae is serious, and their antimicrobial resistance monitoring and resistant mechanism should be strengthened, so as to provide references for the control of the infection spread.