Brucella abortus is an important zoonotic pathogen that causes severe economic loss to husbandry and poses a threat to human health. The B. abortus A19 live vaccine has been extensively used to prevent bovine brucellosis in China. However, A19 has the limitation of diagnosis interference and residual virulence for the host. In this study, a novel genetically marked vaccine, A19ΔvirB12, was generated and evaluated. The results indicated that A19ΔvirB12 was able to provide effective protection against B. abortus 2308 (S2308) challenge in mice and vaccinated sera can be distinguished from infected sera. However, previous studies have found that the accuracy of the serological detection method based on VirB12 protein needs to be improved. Therefore, we attempted to identify potential supplementary antigens with differential diagnosis function using label-free quantitative proteomics. Eighteen proteins identified only in S2308 were screened, and 7 of them were predicted to have high probability of antigenicity. In addition, 12 virulence factors were upregulated in S2308. The upregulation pathways of S2308 were significantly enriched in quorum sensing, ATP-binding cassette transporter, and metabolism. Several proteins related to cell division were significantly downregulated, while some proteins involved in transcription were upregulated in S2308.