Tuberculosis is a particular infectious disease caused by Mycobacterium tuberculosis .The disease primarily affects the lungs and cause pulmonary tuberculosis diagnosed with smear positive. The prospective case-control study was conducted in the Nepal Anti-Tuberculosis Association (NATA), GENETUP lab, Kalimati, Kathmandu from July 2010 to October 2010, on sputum samples from patient visiting at the GENETUP lab with suspected pulmonary tuberculosis cases without treatment and follow-up cases after DOTS and MDR treatment. A total of 299 sputum samples (170 from 78 suspected cases, 42 from 22 follow-up cases with DOTS treatment and 87 from Follow-up cases with MDR) were collected aseptically. The study clearly indicated that the case detection rate (efficacy) of fluorescent microscopy (AO stain) is remarkably higher than that of ZN (light microscopy) ,with aided advantages of less eye strain, easy visualization, less time consuming and even detection of low number of bacteria (paucibacillary cases) in comparison to ZN method. the correct diagnosis of pulmonary tuberculosis requires combination of AO (fluorescent microscopy), culture and biochemical analysis.