Objective To investigate the role of nontypeable Haemophilus influenzae biofilm in children with chronic pulmonary infection and compare the biofilm formation of isolates from acute and chronic pulmonary infection in children. Methods Haemophilus influenzae isolated from bronchoalveolar lavage fluid of acute and chronic pulmonary infection in children was selected from the clinical microbiology lab in Shenzhen Children's Hospital, and Haemophilus influenzae isolated from the nasopharynx of healthy children was selected as the control group. And then we compared the production of biofilms at different time points and between different groups in children with acute or chronic pulmonary infection. Results All strains were nontypeable Haemophilus influenzae detected by PCR of capsular gene bexA. The difference in absorbance between acute and chronic lung infection groups at different time points (days 1, 2, 3, 4, and 7) was statistically significant, and the absorbance was the highest in the chronic infection group on day 4. On the 4th day, the difference in absorbance between the healthy control group and the acute and chronic lung infection groups was statistically significant (P<0.05). The absorbance of the chronic lung infection group was higher than that of the healthy control group and the acute lung infection group (P<0.05). Conclusion The biofilm formation of nontypeable Haemophilus influenzae takes a long time, and the formation ability in children with chronic pulmonary infection is significantly higher than that in children with acute pulmonary infection and healthy control children.