Abstract: Objective To explore the non-bacteria pathogens of acute laryngitis in children. Methods The clinical data and sputum sample were collected from 325 patients hospitalized due to acute laryngitis in consecutive 10 years from January 2006 to December 2015. The multiple non-bacteria pathogens were detected and analyzed with clinical data. Seven types of respiratory viruses were detected by direct immunofluorescence. Mycoplasma pneumoniae (MP), Chlamydia pneumoniae (CP), and Boca virus (HBoV) were detected by fluorescence quantitative PCR. The rhinovirus (HRV) and human metapneumovirus (hMPV) were detected by RT-PCR. Venous blood was collected within 24 h after hospitalization and 7-10 d after treatment. The MP antibody of IgG and IgM were detected by ELISA. Results The detection rate of non-bacteria pathogens was 46.2% in 325 children with acute laryngitis (150/325), including 76 cases (23.4%) of virus and 99 cases (30.5%) of MP. Virus detection rate in 1-3 year old children was obviously higher than in 0-1 year old children and over 3 years old children (χ2 = 9.527, P = 0.009). With the increase of age, the detection rate of MP increased gradually (χ2 = 10.132, P = 0.006). The detection rates of RSV and hBoV were higher in under 3-year-old children. The detection rates of virus in winter and spring were significantly higher than those in summer and autumn (χ2 = 5.064, P = 0.024). The detection rates of MP in winter, spring, summer, and autumn was 13.1%, 25.0%, 38.2%, and 44.9% respectively, and the MP detection rates were increased gradually over seasons (χ2 = 4.438, P = 0.035). The detection rate of RSV was higher in winter, and hBoV was higher in summer. Conclusion Acute laryngitis mainly occurred in children under 3-years-old children, and the detected non-bacteria pathogens were different among different ages and seasons. Virus was the major pathogens in young children, while MP was more common in older children.