Bovine tuberculosis (bTB), mainly caused by Mycobacterium bovis (M. bovis), is a zoonotic disease that poses a significant threat to both cattle and human health. Currently, the immunological diagnosis using tuberculin tuberculin skin testing (TST) is widely employed for bTB surveillance. There is an urgent need for on-farm inspection indicators or strategies for bTB that utilize easily accessible fluid samples. Previously, we presented a comprehensive proteomic overview of plasma-derived exosomes from M. bovis-infected cows. In this study, we used other two parallel cohorts from two farms to illustrate the expression characteristics of infected cows and explore potential bTB indicators using plasma exosome proteome profiling. Cohort A included 15 negative and 22 positive samples, while cohort B included 28 negative and 40 positive samples. Among the 777 identified proteins, 217 in cohort A and 233 in cohort B showed differential expression between negative and positive samples. Of these, 47 proteins exhibited consistent dysregulation in both cohorts and were significantly enriched in tuberculosis, antigen processing and presentation pathways. Additionally, three proteins, HSPA8, B2M and HRG, were identified and verified as bTB indicators through Lasso regression analysis, western blot and ELISA validation. This study enhances us understanding of the dynamic expression change in plasma exosomes following M. bovis infection and provides technical insights for bTB detection based on clinical fluid samples.