ObjectiveInadequate tuberculosis (TB) knowledge and awareness of proper TB control practices among health care workers (HCWs) may increase the risk of nosocomial TB transmission. This study aimed to assess HCWs’ TB-related knowledge and control practices to guide the development of more effective targeted TB health education and training programs.MethodsIn January 2023 a cross-sectional survey was administered to 323 HCWs employed by five primary health care centers and three secondary comprehensive medical institutions in Beijing, China. Survey data were collected using a standard questionnaire.ResultsAnalysis of survey responses revealed TB knowledge and practices awareness rates of 60.4% and 90.6%, respectively. The overall average awareness rate across all 19 TB knowledge- and practice-related questions was 70.0%. Intermediate- and senior-level HCW’s average TB knowledge score was respectively 2.225 and 8.175 times higher than that of primary-level HCWs, while the average TB knowledge score of HCWs in secondary comprehensive medical institutions was 3.052 times higher than that of HCWs in primary health care centers. Higher average TB knowledge score correlated with higher-level professional titles and higher level work units, but higher average TB control practices score correlated with employment at primary health care center rather than secondary comprehensive medical institution. Notably, 13.6% of HCWs had not received TB training during the past three years, while 86.1% expressed willingness to undergo online TB training.ConclusionThese findings highlight inadequate TB knowledge and awareness of proper TB control practices among HCWs in primary health care centers and secondary comprehensive medical institutions in Beijing, underscoring the urgent need for targeted educational and training initiatives to improve TB awareness and control efforts.