Objective To establish a biofilm (BF) models of Haemophilus influenza in vitro, and to observe the changes of antibiotic susceptibility after the BF fromation. Methods Thirty strains Haemophilus influenzae isolated from adenoids of children with adenoidal hypertrophy and cultured in a 96-well plate. The BF was identified by crystal violet staining and scanning electron microscopy (SEM). The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and the minimum biofilm bacteria biofilm clear concentration (MBEC) of ampicillin (AMP), ceftriaxone (CRO), levofloxacin (LVFX) and azithromycin (AZM) were individually detected. Result All of 30 strains of Haemophilus influenzae formed various BF. After BF is formed, the increase of MBEC for different antibiotics was inconsistent with the increase of MIC and MBC. The difference was statistically significant (MBEC/MBC, H=91.54; MBEC/MIC, H=87.91; all P<0.001). The MBEC of AMP was the highest, up to 100 times than the MBC and MIC. The MBEC of CRO was dozens of times than the MBC and MIC. The MBEC of LVFX and AZM were most close to those of MBC and MIC. Conclusion After the formation of BF, resistance to antibiotics of Haemophilus influenzae is enhaced. LVFX and AZM showed more favorable effect on Haemophilus infuenzae BF.