Brucellosis is a vital zoonotic disease caused by Brucella, which infects a wide range of animals and humans. Accurate diagnosis and reliable vaccination can control brucellosis in domestic animals. This study examined novel immunogenic proteins that can be used to detect Brucella abortus infection or as an effective subcellular vaccine. In an immunoproteomic assay, 55 immunodominant proteins from B. abortus 544 were observed using two dimensional electrophoresis (2DE) and immunoblot profiles with antisera from B. abortus-infected cattle at the early (week 3), middle (week 7), and late (week 10) periods, after excluding protein spots reacting with antisera from Yersinia enterocolitica O:9-infected and non-infected cattle. Twenty-three selected immunodominant proteins whose spots were observed at all three infection periods were identified using MALDI-MS/MS. Most of these proteins identified by immunoblot and mass spectrometry were determined by their subcellular localization and predicted function. We suggest that the detection of prominent immunogenic proteins during the infection period can support the development of advanced diagnostic methods with high specificity and accuracy; subsidiarily, these proteins can provide supporting data to aid in developing novel vaccine candidates.