Background: Despite the advances in the diagnosis of tuberculosis, effective diagnosis remains a challenge among children. Several countries have rolled out effective diagnostic methods but capacity of health care workers to achieve expected resultshave not been evaluated. The objective of this study was therefore to assess the knowledge, practices and perception of health care workers on diagnosis of childhood tuberculosis at public and private health facilities in Bureti sub-county, Kenya. This is important for effective diagnosis hence reduced childhood deaths. Methods:A cross-sectional study was carried out on44 health care workers in 10TB clinic in both public and private health facilities. The data collection tools were key informant guide and semi-structured questionnaire. Results:Majority of Health care workers(45.5%)have poor knowledge,with 31.8%mentioning that mycobacterium leprae also causes TB. On practices, 68% confirms that gene expert is available in the health facilities but only 30% can interpret its results and 20% do not know how to use it. Majority of the respondents (61.4 %) believe stigma still remain a challenge in diagnosis of TB. Conclusion:Knowledge, practices and perception need to be improved among the health care providers for better diagnosis of childhood tuberculosis. There is a need for regular training on diagnostic methods and more importantly interpretation of results. In addition, there is a need for health education so address the challenges of community stigma. Also, the study recommends more studies in other counties to inform a national strategy for effective diagnosis of childhood TB.