Bovine tuberculosis (bTB), primarily caused by Mycobacterium bovis (M. bovis), is a globally zoonotic disease with significant economic impacts. Plasma exosomes have been extensively used for investigating disease processes and exploring biomarkers. While mass spectrometry (MS)-based proteomic analysis of plasma exosomes has been employed for human tuberculosis (TB) studies, it has not yet been applied to bTB. Therefore, a comprehensive proteomic overview of plasma exosomes from M. bovis-infected cows is essential. In this study, we presented an extensive proteomic analysis of plasma exosomes from 89 M. bovis-infected cows across three farms, using data dependent acquisition (DDA) mode. Our analysis encompasses 239,894 spectra, 6,011 peptides and 835 proteins. The proteomic overview revealed both consistencies and differences among individual cows, supplements 595 proteins to the bovine exosome library, and enriches tuberculosis and related pathways. Additionally, six pathways were validated as immune response pathways, and three proteins (CATHL1, H1-1, and LCN2) were identified as potential indicators of bTB. This study is the first to investigate the exosome proteome of plasma from cows infected with M. bovis, providing a valuable dataset for exploring candidate bTB markers and understanding the mechanisms of host defense against M. bovis.