[Objective] Brucellosis (Brucellosis) referred to as cloth disease, is caused by Brucella to livestock-based zoonotic infectious diseases, causing serious public health problems. At present, the main method of eliminating the disease around the world is the combination of culling and immunization, so the establishment of rapid and accurate diagnostic methods for the prevention and removal of brucellosis is necessary. We establish a diagnostic method of fluorescence polarization (FPA) for brucellosis and provide a scientific and efficient method for diagnosis of brucellosis. [Methods] In the present study, the conjugate of lipopolysaccharide O-chain (OPS) and fluorescein isothiocyanate (FITC) was purified from purified S2 strain of Brucella spp. As an antigen, and the optimal dilution, dilution concentration, reaction conditions, the results to determine parameters such as the initial establishment of the Brucella fluorescence polarization diagnosis method. The results showed that the sensitivity and specificity of the positive serum of 148 bovine serum (including 70 bovine serum and 78 goat serum) and 155 negative bovine serum (including 82 bovine serum and 73 goat serum) were determined by this method. The intra-assay and inter-assay reproducibility was assessed using the controlled positive and negative serum assay kits. At the same time, 400 samples of bovine serum samples were detected by FPA kit and commercial kit, and the coincidence rate was compared. [Results] The optimal conditions of each component in the kit were: the best sample dilution was 0.5% sucrose phosphate buffer; the concentration of the labeled antigen was 90 μg/mL; the best reaction time was 3 to 5 minutes; δmP value of <20, was negative, δmP value of ≥20, was positive. The sensitivity of the method was 98.6% and the specificity was 98.7%. The comparison of 400 clinical samples showed that the FPA method established in this study coincided with the import commercialization kit 94.0%. [Conclusion] The method of fluorescence polarization diagnosis with good specificity and sensitivity was established.