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Bovine tuberculosis is usually diagnosed using tuberculin skin tests or at post-mortem. Recently, we have developed a serological test for bovine tuberculosis in cattle which shows a high degree of accuracy using serum samples. Here, we have assessed the performance of the test using individual bovine milk samples. The diagnostic specificity estimate using the high sensitivity setting of the test was 99.7% (95% CI: 99.2–99.9). This estimate was not altered significantly by tuberculin boosting. The relative sensitivity estimates of the test using the high sensitivity setting in milk samples from comparative skin test positive animals was 90.8% (95% CI: 87.1–93.6) with boosting. In animals with lesions, the relative sensitivity was 96.0% (95% CI: 89.6–98.7). Analysis of paired serum and milk samples from skin test positive animals showed correlation coefficients ranging from 0.756–0.955 for individual antigens used in the test. Kappa analysis indicated almost perfect agreement between serum and milk results, while McNemar marginal homogeneity analysis showed no statistically significant differences between the two media. The positive and negative likelihood ratio were 347.8 (95% CI: 112.3–1077.5) and 0.092 (95% CI: 0.07–0.13) respectively for boosted samples from skin test positive animals. The results show that the test has high sensitivity and specificity in individual milk samples and thus milk samples could be used for the diagnosis of bovine tuberculosis. [ABSTRACT FROM AUTHOR]