Prion diseases, caused by proteinaceous infectious particles, are a group of rare and fatal neurodegenerative disorders with high interspecies transmissible risk which has no cure. Prion diseases do not have a definitive diagnostic marker and pose a serious threat in the lives of humans and animals. The threat from the disease is aggravated due to long incubation periods of 2-4 decades that has led to the demand for early biomarkers. The aim of this study was to identify miRNA biomarkers expressed in tissue from a prion infected organotypic brain slice model including extracellular vesicles secreted into the conditioned media. This research involved optimising a method to isolate exosomes from a very small volume of conditioned medium that is used to culture the organotypic brain slices. The seven significantly expressed miRNAs (mmu-miR-124-3p, mmu-miR-218-5p, mmu-miR-128-3p, mmu-miR-195a-5p, mmu-miR-29c-3p, mmu-miR-107-3p and mmu-miR-138-5p) with greater than 2-fold down-regulation in the terminal stage prion infected brain slice model tissue were determined using small RNA deep sequencing. One miRNA, mmu-miR-29c-3p, was found to be significantly differentially expressed in both the pre-clinical and clinical stage of the prion brain model used in this study. Moreover, exosomes secreted from the prion organotypic brain model were also profiled to identify potential prion exosomal miRNAs that could be used as peripheral biomarkers. Additionally, this study also demonstrates that neuronal exosomes, loaded with miRNA, can be taken up by brain endothelial cells, potentially translocate through the blood-brain barrier and serve as a diagnostic source of blood based biomarkers for neurodegenerative diseases.