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Background: Human brucellosis is a neglected and zoonotic disease also known as Malta fever or undulant fever. Objectives: A cross sectional study was conducted to determine the seroprevalence of brucellosis and to access the role of risk factors associated with this disease in humans of Punjab, Pakistan. Methods: A total of 250 serum samples were collected and subjected to Rose Bengal Plate Test and Enzyme Linked Immunosorbent Assay for screening of Brucella. A predesigned questionnaire was filled prior to sampling to collect data regarding socio -demographic and suspected risk factors of human brucellosis. Descriptive and bivariate statistical analysis was performed using the STATA software version 12. Results: The study showed 16% seroprevalence of brucellosis. The prevalence was statistically higher in males (24%), age group of 20 to 30 years (26.92%), rural residents, (23%) and individuals with animals at home (22.50%). Among the related risk factors, exposure to animals (OR = 1.87, 95% CI: 0.9459, 3.6973) and consuming raw milk (OR = 2.36, 95% CI: 1.1713, 4.7760) were strongly associated with the disease. Conclusions: Awareness programs in the rural population should be provided about the disease and its associated risk factors. Consuming unpasteurized milk and products should be avoided to control this disease.