The objective of the present study was to investigate the usefulness of a recombinant flagellar protein, FlaB, of Leptospira interrogans serovar pomona in the serodiagnosis of leptospirosis by the fluorescence polarization assay (FPA). The recombinant protein FlaB was purified to homogeneity by a combination of nickel-nitriloacetic acid agarose chromatography, electrophoresis, and electroelution. Purified FlaB was labeled with fluorescein isothiocyanate (FITC). Western blotting was performed by using bovine sera with microscopic agglutination test (MAT) titers of antibodies against L. interrogans serovar pomona and L. bergpetersenii serovars hardjo and sejroe to confirm the antigenicity of FlaB. Western blot analysis demonstrated that labeled as well as unlabeled FlaB was recognized by the positive sera tested, indicating the broad serovar cross-reactivity of this protein. It also indicated that labeling with FITC did not affect the antigenicity. By using FITC-labeled FlaB as a tracer antigen, a homogeneous FPA was developed to detect antileptospiral antibodies in bovine sera. A population of 208 MAT-positive and 208 MAT-negative serum samples was tested by FPA. The FPA cutoff was determined by receiver operating characteristic analysis. By FPA, 83.7% of the MAT-positive serum samples were positive and 81.2% of the MAT-negative serum samples were negative. Compared to the results of MAT, the positive predictive value of FPA was 81.7% and the negative predictive value of FPA was 83.3%. The FPA is a simple and rapid technique for the detection of anti-Leptospira antibodies.