Update information. Biliary tract infection (BTI) is a common abdominal disease and without proper management can even lead to bacteremia and further death. Although studies have focused on the initiation and mechanisms of this disease, diagnostic biomarkers for BTI are still lacking. Extracellular vesicles (EVs) are a kind of biological noano-particles that released from multiple types of cells reflected different physiological or pathological conditions. In the past decades, the development of EV-based diagnostic methods for many diseases is emerging. However, the utilization of EV-directed approaches for BTI diagnosis has not been well-investigated. To explore effective protein biomarkers for patients with BTI, we applied a label-free quantitative proteomic method, which is unbiased and high through-put, on the EVs from serum of control healthy donors or patients with BTI in this study. After analyzing the results, 62 novel proteins were unique in the BTI samples from the healthy controls. Moreover, 192 differentially expressed proteins were identified in EVs from patients with BTI when compared with those from healthy controls. To gain an overview on the functions of these proteins and the biological processes/pathways that the proteins are involved in, we performed GO and KEGG analysis by using this set of proteins an input. Importantly, some key pathways involved in fat acid processing were enriched as key pathways. Here we first idenfified biomarkers, Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) and Crumbs homolog 3 (CRB3) presented significant different between BTI patients and healthy individuals. Clinical validation from plasma derived EVs samples that demonstrated its specificity to BTI. Our work applied an unbiased proteomic tool to identify CEACAM1 and CRB3 protein biomarkers in serum EVs may facilitate the development of diagnostic systems to identify patients with BTI.