Background: Childhood tuberculosis is a major global public health problem and is responsible for a considerable burden of overall tuberculosis disease. It accounts for 6–10% of all tuberculosis cases worldwide. Primary health care workers are the first contact of the community to the healthcare delivery system and they attend to large populations in the community. Hence, this study assessed knowledge and case detection practices of childhood tuberculosis among these workers in Osun State. Methodology: A descriptive cross–sectional study was conducted using a multi-stage sampling technique to recruit 220 respondents. A semi–structured pretested self-administered questionnaire adapted from similar previous studies was used to collect data. Data were entered into IBM SPSS version 25 after correction for errors. Bivariate analysis was done using Chi-square and multivariate logistic regression was used to identify the predictors of knowledge and case detection practice at 5% level of significance. Result: Forty-seven (21.4%) respondents had good knowledge of childhood tuberculosis and 181 (82.3%) had good case detection practice. The predictor of good knowledge of childhood tuberculosis among respondents was previous participation in childhood tuberculosis training (AOR=7.919, p < 0.001) while predictors of good case detection practices among respondents were direct involvement in tuberculosis control (AOR=4.405, p = 0.002) and previous participation in childhood tuberculosis training (AOR=4.726, p<0.014). Conclusion: The study highlights the poor knowledge of childhood tuberculosis. Hence, specific trainings on childhood tuberculosis are urgently needed among PHC staff in Osun State, Nigeria