BACKGROUND: Brucella spp. are Gram-negative bacteria causing brucellosis, a major zoonotic disease affecting animals and humans. Annually, over 500,000 human cases are reported globally, with many undiagnosed due to nonspecific symptoms and diagnostic challenges. Current methods for Brucella detection, such as culture and serology, are time-consuming and lack specificity, hindering effective disease control. This study aims to develop a novel dual priming oligonucleotide (DPO) system-based PCR method for the specific detection of Brucella spp. in slaughter-aged livestock. This approach provides a rapid, sensitive, and field-deployable tool to improve early diagnosis and control of brucellosis. METHODS: We developed a DPO system-based PCR assay for the specific detection of Brucella spp. in slaughter-aged livestock. The method utilizes two sets of primers designed to specifically target unique regions of the Brucella genome. The assay was validated for specificity using a panel of 15 non-target bacterial species commonly found in livestock, including Escherichia coli, Salmonella spp., and Campylobacter spp. Sensitivity was evaluated using DNA extracted from a range of Brucella strains, with detection limits assessed using serially diluted samples. The assay's performance was further tested on 500 samples from slaughter-aged sheep to assess its applicability in field conditions. RESULTS: The DPO-based PCR assay demonstrated excellent specificity, with no cross-reactivity observed in any of the 15 non-target bacterial species tested. The assay was able to detect Brucella spp. at low DNA concentrations, with a sensitivity limit of approximately 5.3 × 10 CONCLUSIONS: The DPO-based PCR assay provides a highly specific, rapid, and cost-effective tool for the detection of Brucella spp. in slaughter-aged livestock. This method is suitable for routine surveillance in slaughterhouses, offering a promising solution for early detection and control of brucellosis in both livestock and public health contexts. The assay's simplicity and robustness make it an ideal candidate for field deployment, particularly in resource-limited settings where timely disease control is crucial. CLINICAL TRIAL NUMBER: Not applicable.