Objective: To investigate the knowledge and practice levels on infectious disease among primary and middle school students, so as to provide a basis for targeted infectious disease in health education and infectious disease prevention and control in schools. Methods: The stratified cluster random sampling was used to select 25 670 primary and middle school students from 6 urban area and 10 suburban area in Beijing from October to November in 2020. A questionnaire survey on knowledge and practice of students related to infectious disease prevention and control was conducted using the questionnaire compiled by the Chinese Center for Disease Control and Prevention. Chisquare test was used for group comparison. Results: During the past two weeks, 8.64% students reported sickness absence. The awareness rates of knowledge on infectious disease were 37.64%-93.56%, and the formation rates of practice on infectious disease were between 62.22%-98.74%. The overall awareness rate of knowledge on infectious diseases was 58.58%, and the overall formation tate of practice on infectious disease prevention was 78.19%, with boys (54.14%,73.67%) lower than girls (63.25%,82.94%),urban areas (62.88%,79.12%) higher than suburbans areas (55.30%,77.48%), vocational high school studengs (43.51%,68.13%) lower than nonvocational high school students (elementary school: 56.05%, 85.19%; middle school: 61.66%, 78.63%; high school: 66.72%, 72.77% ), residential students (55.21%,71.59%) lower than nonresidential students (59.32%,79.64%), students living with parents (59.22%,79.30%) higher than those not with parents (54.60%,71.34%), and the differences were statistically significant (χ2=219.08, 148.95, 514.34, 26.44, 27.12; 323.26, 9.91, 536.11, 144.32, 114.11, P<0.05). Conclusions The levels of knowledge and practice regarding relative infectious disease among primary and middle school needs to be improved. Health education on the weak links in knowlege and practice of infectious disease prevention and control should be strengthened to reduce the incidence and transmission of infectious diseases.