Objective: To understand the trends and associated factors of tuberculosis knowledge and behavior among freshmen from Jiangsu Province, 2019 to 2022, so as to provide theoretical support for the popularization of tuberculosis knowledge and the prevention and control of tuberculosis in universities. Methods: From 2019 to 2022, a total of 33 944 freshmen from 20 universities in Jiangsu Province were selected by stratified random sampling, and tuberculosis knowledge and behavior were surveyed online using a selfdesigned questionnaire. Oneway variance trend test and multiple linear regression analysis were used to analyze the trend of knowledge and behavior levels, and a multiple linear regression model was used to analyze the influencing factors. Results: The overall awareness rate of tuberculosis among freshmen from Jiangsu Province was 90.6%, and the overall awareness rates from 2019 to 2022 were 88.4%, 90.4%, 91.7%, and 91.8%, respectively. The overall behavioral accuracy rate was 92.3%, with the annual rates from 2019 to 2022 being 91.8%, 92.3%, 92.7%, and 92.4%, respectively. Both the awareness rate and accuracy behavior rate increased annually (F=216.67, 11.75, P<0.01). The multiple linear regression analysis showed that school type, ethnicity, fathers educational level, mothers educational level, mothers occupation, family per capita monthly income, and living arrangement in high school affected both personal knowledge awareness rate and personal behavioral accuracy rate (β=-0.047-0.035, P<0.01), while region, gender, family residence, and being an only child only affected behavioral accuracy rate (β=-0.003-0.032, P<0.05). Conclusions The tuberculosis knowledge and behavior levels of freshmen from Jiangsu Province have been increasing by year. Targeted health education should be carried out based on the relevant influencing factors to further enhance the tuberculosis prevention and control literacy.