Recently, microRNAs (miRNAs) have been implicated in regulating neuroinflammatory and demyelinative responses in multiple sclerosis (MS) and its mouse model of experimental autoimmune encephalomyelitis (EAE). miRNAs have also been studied as biomarkers of disease pathology and drug-response in MS. However, no complete miRNA profiling at various stages of EAE disease has been examined, especially in the urine. We carried out a systematic analysis of miRNAs in the urine exosomes as well as in the plasma and spinal cord at pre-onset, onset and peak stages of EAE established in the chronic B6 mice model. For the first time, we provide evidence that urine exosomes can be a specific and sensitive source of miRNA biomarkers for all 3 stages of EAE disease. In a significant observation, we observed that miR-155-5p expression increased in urine exosomes, plasma and spinal cord 6 days before the onset of disease, suggesting its early involvement in the pathology of EAE disease. We also analyzed the effect of Glatiramer acetate (GA; copaxone) treatment, an approved treatment for MS patients, in modulating miRNA expression at the peak of EAE disease. We identified miR-155-5p, miR-27a-3p, miR-9-5p and miR-350-5p as putative GA-treatment responsive miRNA biomarkers. Since, EAE is a mainly CD4 cells mediated disease, we also examined the above set of miRNAs and found to be significantly altered in T cells polarized to Th1 and Th17 phenotype, similar to urine exosomes. Thus, urine exosome miRNAs hold the potential to be defined as novel accessible stage-specific biomarkers of EAE (MS) disease as well as treatment response.