Edible plant derived exosome-like nanoparticles (ELNs) have been shown to have multiple nutraceutical functions. However, the diversity of plant materials makes the plant derived ELN study challenging. More efforts are still needed to explore the feasible isolation methods of edible plant derived ELNs and the possible roles of food-derived ELNs in improving human health. In this study, a size exclusion chromatography based method was compared with the traditional ultracentrifugation method to isolate blueberry derived ELNs (B-ELNs), and the miRNA profile of B-ELNs was analyzed by high-throughput sequencing. A total of 36 miRNAs were found to be enriched in B-ELNs compared with berry tissue, and their potential cross-kingdom human gene targets were further predicted. Results showed that size exclusion chromatography was effective for B-ELN isolation. The most abundant miRNAs in B-ELNs mainly belonged to the miR166 family and miR396 family. Target gene prediction indicated that B-ELNs could potentially regulate pathways related to the human digestive system, immune system and infectious diseases.