Objective: The World Health Organization (WHO) has recommended to consider the early morning sputum samples test instead of the conventional two days for the diagnosis of smear positive tuberculosis (TB). Materials and Methods: This hospital-based cross sectional, comparative study was conductedat Medical College Hospital, Kolkata during November 2018 to May 2020.Two sputum specimens were collected from enrolled presumptive TB patients. In addition, a second sample was collected by traditional approach. All the samples were examined by Ziehl-Neelsen staining. Chi square testwas used to compare statistical differences in the proportion smear positive between the two approaches. Results: Total 230 patients were evaluated in the present study. The majority of patients (47.4%) belonged to the age group of 41-60 years, followed by ≤40 years age group (41.3%) and ≥61years age group (11.3%).Among 230 presumptive TB patients, 73 patients were detected as sputum positive presumptive TB (PTB) patients. High bacillary load (3+) was documented in conventional morning sputum compared to first day sputum (14.1% vs. 13.8%) and same day second sputum (14.1% vs. 7.4%). Total 71 patients were detected and 2 patients (2.7%) were missed on conventional sputum testing whereas, on the same day sputum testing, 67 patientswere smear positive while sixpatientswere found to bemissed. Conclusions: The conventional sputum microscopy method is more sensitive than same day method.