Background & Objectives: Brucellosis remains an important occupational zoonotic disease, especially in developing countries. The disease is endemic in Iran and the Fars province. One of the main routes of brucellosis infection is at slaughterhouses, where the workers directly contact infected animals. This study was designed to estimate the seroprevalence of brucellosis among slaughterhouse workers in the Fars province, Iran. Materials & Methods: Ninety blood samples were collected from workers of two livestock slaughterhouses (Marvdasht and Kazeroon), in Fars, Iran. The sera were assessed for the Rose Bengal test (RBT), as a screening test for brucellosis, and the positive samples were subjected to the Wright test. The positive Wright samples were finally tested for the 2 -mercaptoethanol (2 -ME) agglutination test. Results: Brucellosis prevalence was 13.33% using RBT and 4.44% of the workers showed active brucellosis. No significant relationship was found between the questionnaire variables and brucellosis tests; exceptionally, there was a relationship between the workers' statements regarding having had brucellosis and RBT (P=0.01). Conclusion : Our study highlights the practical application of serological tests, including RBT, Wright, and 2 - ME as a simple strategy to monitor brucellosis and to diagnose and treat its active form in endemic regions. Although a small frequency of the disease was found, it could cause significant health and economic damage to humans and animals in endemic areas. Furthermore, taking enough protective measures is highly recommended for slaughterhouse workers to prevent human brucellosis.