Introduction. Brucellosis is prevalent in Mediterranean countries. The aim of this study was to determine the seroprevalence of brucellosis and associated factors among Syrian refugees in Jordan. Methods. A cross-sectional study was conducted among adult Syrian refugees who attended the Public Health Lab (PHL) in Al Mafraq governorate, during the period of May-June 2022 to obtain a health certificate, which is legally required to receive governmental authorization for employment in Jordan. Blood samples were obtained from participants and a serum specimen was tested for the presence of IgG antibodies against Brucella using enzyme-linked immunosorbent assay (ELISA) IgG kits (Vircell Microbiologists, Granada, Spain). Results. A total of 1562 Syrian refugees were enrolled in the study. Their ages ranged between 18 and 74 years, with a median age of 30 years at presentation. The majority were males (75.9%, n = 1186) and 24.1% (n = 376) were females. The Brucella ELISA IgG results were positive for 149 persons, with an overall seroprevalence rate of 9.5% (95% confidence interval: 8.0%–11.0%). Having animal-related occupations, residing outside refugee camps, consuming unpasteurized milk, handling animals or their tissues, and slaughtering animals within 6 months of study inclusion were significantly higher among the seropositive group. In the multivariate analysis, IgG-positive persons were 13 times more likely to report being diagnosed with brucellosis (OR = 13.1, 95% CI: 6.1–28.3; p ≤ 0.001). In addition, they were more likely to reside in the city of Al Mafraq, as opposed to a refugee camp (OR = 1.9, 95% CI: 1.1–3.2; p = 0.025) and to have handled animals within 6 months of study inclusion (OR = 3.1, 95% CI: 1.1–8.9; p = 0.035). Conclusions. In conclusion, one-tenth of adult Syrian refugees were tested positive for Brucella ELISA IgG. Being diagnosed with brucellosis, residing in the city of Al Mafraq, as opposed to a refugee camp, and handling animals within 6 months of study inclusion were significantly associated with being positive for Brucella ELISA IgG. This study illustrates the need for improved brucellosis control measures via comprehensive vaccination of animals and enhanced laboratory detection and surveillance capacities, in addition to emphasizing the need for increased awareness sessions among Syrian refugees on the safe use and preparation of dairy products and safety practices of handling animals and their tissues.