Objective: To understand knowledge and practice of influenza prevention and associated factors among primary and middle school teachers, and to provide a reference for conducting the relevant propaganda work of influenza. Methods: Stratified random cluster sampling method was used to select 858 primary and secondary school teachers from Huainan of Anhui Province to complete questionnaires on influenza prevention. Results: Television and radio broadcasting were the basic tools for teachers to acquire flu knowledge, with 74.12% and 80.00% of primary and secondary school teachers, respectively. The total awareness rate of influenza knowledge among primary and middle school teachers was 56.63% and 58.63%, respectively. The results of univariate analysis showed primary and secondary school teachers’ influenza awareness were significantly affected by regions and education levels. In addition, primary school teachers’ influenza awareness was significantly affected by full-time medical technicians or part-time medical workers, secondary school teachers’ influenza awareness was significantly affected by working years(P<0.05). The total formation rates of influenza-related health behavior between primary and secondary school teachers were 71.20% and 73.00%, respectively. The results of univariate analysis showed primary and secondary school teachers’ influenza health behaviors was significantly affected by regions, educational levels, full-time medical technicians or part-time medical workers and health training. Moreover, secondary school teachers’ influenza health behaviors was significantly affected by working years(P<0.05). Multivariate logistic regression analysis showed that whether the comprehensive scores of influenza knowledge and behavior were qualified was correlation with the regional and educational levels. Additionally, the factors whether it is a full-time medical technician or a part-time medical teacher also had an effect on the comprehensive score of behavior(P<0.05). Conclusion The influenza knowledge level and the health behavior formation rate in the primary and secondary school teachers need to be improved, the knowledge of influenza and the relevant influencing factors should be taken into consideration to take targeted health intervention measures to improve their ability to fight against influenza.