Exosomes have been identified as important carriers of various genetic materials, including microRNAs (miRNAs). Increasing evidence indicates that the course of severe acute pancreatitis (SAP) is associated with miRNAs transported by exosomes. We aimed to identify the signature miRNAs as biomarkers of SAP.We obtained exosomes from the SAP patients' blood. After separation, purification, and identification, we performed high-throughput sequencing and screened the differentially expressed(DE) miRNAs in the exosomes. Bioinformatics analysis was performed to identified the target genes of the miRNAs and the pathways enriched based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses, and selected the key miRNAs related to SAP. Total RNA was extracted from patient serum exosomes to detect the expression levels of the selected miRNAs in exosomes of three experimental groups (mild -, moderately severe -, and severe AP) and a control group, using Real-time quantitative polymerase chain reaction (RT-qPCR).272 DE miRNAs were identified between SAP and control group. Using bioinformatics analysis, we determined that the functions of the target genes were enriched in six signaling pathways including focal adhesion. Based on this, seven candidate signature miRNAs were selected: miR-603, miR-548ad-5p, miR-122-5p, miR-4477a, miR-192-5p, miR-215-5p, and miR-583. The RT-qPCR results of the seven miRNAs in the SAP group were consistent with the sequencing results.Exosome-derived miR-603, miR-548ad-5p, miR-122-5p, miR-4477a, miR-192-5p, miR-215-5p, miR-583 are positively correlated with SAP, which might provide new insights into the pathogenesis of SAP and serve as the biomarkers of SAP.Copyright © 2022 Asian Surgical Association and Taiwan Robotic Surgery Association. Published by Elsevier B.V. All rights reserved.