This study was performed to determine the seropositivity of human brucellosis among the patients suffering from pyrexia of unidentified origin. This cross-sectional study was performed at department of Microbiology, Mymensingh Medical College, Mymensingh, Bangladesh from September 2018 to August 2019; among the patients of pyrexia of unknown origin visited inpatient and outpatient facility of department of Medicine and department of Paediatrics, Mymensingh Medical College Hospital (MMCH) in Mymensingh division of Bangladesh. A total of 400 serum samples were screened by Brucella-specific latex agglutination test to determine seropositivity. Seven percent (7.0%) (28/400) serum samples were found to be seropositive for brucellosis by detecting Brucella-specific antibody at a titer ≥1:160. Therefore, Brucella-specific latex agglutination test may be recommended as a screening test for human brucellosis in developing and underdeveloped countries.