Inadequate knowledge of tuberculosis can affect people’s attitude and practice, leading to health-seeking delay, treatment default and discrimination against tuberculosis patients. The objective of this study was to assess the knowledge, attitude and practice of tuberculosis among new tuberculosis patients and nontuberculosis patients, and explore the differences of influencing factors between the two groups. Between June 2013 and December 2014, a survey about the knowledge, attitude and practice of tuberculosis was carried out in Anqing, China. Data were collected using a structured questionnaire (intervieweradministered questionnaire for illiterate participants and self-administered questionnaire for literate participants) and tuberculosis management system. Non-conditional logistic regression was used to determine the influencing factors of tuberculosis knowledge. The median of tuberculosis knowledge score was 12 [interquartile range: 5-17] for new tuberculosis patients and 9 (interquartile range: 4-15) for non-tuberculosis patients. In terms of correct answers of tuberculosis knowledge, there were significant differences of tuberculosis detection methods, some items of tuberculosis transmission and main tuberculosis infectious source between new tuberculosis patients and non-tuberculosis patients. Out of the eight items of attitude and practice, only two items had significant differences between new tuberculosis patients and non-tuberculosis patients. The influencing factors of new tuberculosis patients’ knowledge were also different from non-tuberculosis patients’. The more efforts should be made to improve their knowledge and change their attitude and practice towards tuberculosis.