Prion diseases are transmissible neurodegenerative disorders distinguished by long pre-clinical incubation periods during which the infectious prion actively propagates in the brain and other tissues. We previously demonstrated that exosomes assist in the propagation of prions and that the exosomal RNA contents can be used as biomarkers of prion infection. This study aims to determine whether there is clinical utility of miRNA biomarkers during pre-clinical and clinical disease. To define miRNA profile changes over the incubation period, a time-course using a mouse-adapted model of human prion disease and next-generation sequencing were employed to profile miRNA associated with prion infection. Thalamus brain sections and serum samples were collected at 3 and 13 weeks post-inoculation, representing the early and late pre-clinical stages of the disease. Tissues at the clinical stage were also collected following signs consistent with terminal prion disease. Profiling of miRNA expression revealed a collection of miRNAs that are differentially expressed during the development of prion disease in this model. These results were validated in human clinical samples and in an organotypic brain slice model. Prion associated miRNAs identified in the thalamus tissue were also present in extracellular vesicles isolated from serum across each time-point demonstrating potential clinical utility. This study has discovered clinically relevant miRNAs that may benefit the progress of diagnostic development to detect prion-related diseases such as CJD.