Alzheimer's disease (AD) is the most common neurodegenerative disease in the older adults. Although much effort has been made in the analyses of diagnostic biomarkers, such as amyloid-β, tau, and neurofilament light chain, identifying peripheral blood-based biomarkers is in extremely urgent need for their minimal invasiveness and more convenience. Here we characterized the miRNA profile by RNA sequencing in human serum exosomes from AD patients and healthy controls (HC) to investigate its potential for AD diagnosis. Subsequently, Gene Ontology analysis and pathway analysis were performed for the targeted genes from the differentially expressed miRNAs. These basic functions were differentially enriched, including cell adhesion, regulation of transcription, and the ubiquitin system. Functional network analysis highlighted the pathways of proteoglycans in cancer, viral carcinogenesis, signaling pathways regulating pluripotency of stem cells, and cellular senescence in AD. A total of 24 miRNAs showed significantly differential expression between AD and HC with more than ± 2.0-fold change at p value < 0.05 and at least 50 reads for each sample. Logistic regression analysis established a model for AD prediction by serum exosomal miR-30b-5p, miR-22-3p, and miR-378a-3p. Sequencing results were validated using quantitative reverse transcription PCR. The data showed that miR-30b-5p, miR-22-3p, and miR-378a-3p were significantly deregulated in AD, with area under the curve (AUC) of 0.668, 0.637, and 0.718, respectively. The combination of the three miRs gained a better diagnostic capability with AUC of 0.880. This finding revealed a miR panel as potential biomarker in the peripheral blood to distinguish AD from HC.