Back ground: Tuberculosis is a major global health problem, Ethiopian ranks th among 22 high burden countries. Common Laboratory diagnosis methods of tuberculo­ sis are Ziehl-Neelsen light microscopy; light emitting diodes fluorescent microscopy in resource limited countries like Ethiopia, however, detection of TB require skilled labora­ tory personnel, continuous monitoring and quality improvement mechanisms. Objective: To evaluate the performance of laboratory professionals and associated factors on TB microscopy Method: Institutional based cross sectional study was conducted, from Nov 5-30, 2015; 276 laboratory professionals were included in the study. Data were collected using panel testing slide and questioner. Variables which showed association at p < 0.2 in the bivariate analysis were fitted to the multivariate binary logistic regression model. OR with 95% Cl & P-value< 0.05 was used to measure association in multivariate binary logistic regression. Results: Of the respondents, 216 (78.3%) performed TB microscopy correctly; (95% Cl: 73.2, 83.3); while 60(21.7%) participants committed major error. Only 3(1.1 %) of the participants read all (10) slides exactly without any type of error. Those who participa­ tion in decision making about laboratory issues in health facilities perform TB microsco­ py correctly 60% times less likely than those who do not (AOR=0.38,95% Cl (0.19, 0.77). participants who involve in external Quality Assurance performers 2.07 times more likely to perform TB microscopy as compared with those who do not EQA(AOR=2.07,95% Cl (1.01,4.20). Respondents who used Standard Operating Pro­ cedure while performing TB diagnosis in the laboratory had 9.59 times more likely to di­ agnose TB correctly as compared to those who don't use SOP(AOR=9.59,95%CI (2.45,37). Males are 2.86 times more likely to diagnose TB microscopy correctly than females (AOR=2.86, 95%