This prospective study was evaluated the role of TH agglutinins and reevaluate the diagnostic value of Widal test in the diagnosis of typhoid fever by identifying the S. typhi specific antibody. The study was carried out in the department of Microbiology, Mymensingh Medical College, Mymensingh between July 2010 and June 2011, including 200 individuals of different ages and sexes. Widal test and blood culture were performed for each of the cases and controls. Among 150 blood samples from the suspected cases, 23(15.3%) were culture positive for S. typhi and 67(44.7%) had high titres of ≥1:160 for 'O' or 'H' or both agglutinins. Out of 23 culture positive cases, 3(13%) had TO ≥:160, 5(21.7%) had significant TH ≥1:160. Based on the above findings, it was concluded that judicious interpretation of Widal test play an essential role in diagnosis of typhoid fever, especially in the resource poor countries like Bangladesh.