Background Tuberculosis (TB) is a major public health problem in Bhutan despite the implementation of directly observed treatment short-course since 1997. This study aimed to assess the knowledge, attitude and practice on TB among the teacher trainees of Samtse College of Education. Methodology A cross-sectional study was conducted among the teacher trainees of Samtse College of Education. A standardized pretested questionnaire was distributed and self-administered. The participants were selected randomly using randomization. The data was entered in Epi-data 188.8.131.52 and analysed using STATA version 13. A score of 1 point for a correct answer and 0 for wrong/don't know answer was given. The knowledge, attitude and practice score were divided into good and poor based on 50% cut off. Logistic regression was used for the analysis to identify the significant covariates. Results A total of 420 trainees responded to the survey questionnaire. The average knowledge score on TB was 10.7 (Range = 0-21). Two hundred and forty respondents (58.6%) had low knowledge (mean score 7.8±2.5) on TB. Cough, chest pain and weight loss were correctly reported as the symptoms of TB by 306 (72.9%), 187(44.5%) and 187 (44.5%) participants. Eighty-nine-point five percent (376) of participants reported coughing as the main route of TB transmission and 85% (357) of the participants said that it could be prevented by covering the mouth while coughing. In multivariable analysis; the trainees in the junior years of college had good knowledge of TB compared with the senior years (adjusted odds ratio [AOR] 2.02; 95% confidence interval [CI] 1.18-3.5; p-value 0.011). Respondents previously treated for TB were more likely to have good knowledge on TB compared to those who never had TB in the past (AOR 2.39; 95% CI 1.07-5.31; p-value 0.033). The majority (93%) of respondents had a good attitude towards TB cases. Female trainees were 2.4 (95% CI 1.02-5.62; p-value 0.045) times more likely than male trainees to have a positive attitude towards TB. Eighty-eight percent of the respondents reported that they would visit the hospital if they had TB symptoms. The mean score for the practice on TB was 1.33±0.59 (Range:0-2). Conclusion In this study, the majority of the trainees had poor knowledge on TB, especially among the trainees in senior years of college and those who had never suffered from TB. The attitude towards TB was good especially among the female trainees. However, the overall practice was poor among the participants. Therefore, the Ministry of Health should collaborate with relevant stakeholders especially the Ministry of Education to incorporate topics on TB in the syllabus of students and colleges to create awareness on it.