MicroRNAs (miRNAs) are crucial to the immunopathogenesis of multiple sclerosis (MS). The mechanism of action of interferon beta (IFN-β) in relapsing-remitting (RR) MS patients is largely unknown. miR-145 and miR-20a-5p previously reported as diagnosis biomarker in treatment naïve RRMS patients and their expression after IFN-β therapy might be indicative of molecular mechanism of IFN-β. Cross-talking between JAK/STAT pathway and complementary pathways like MAPK is important in IFN-β signaling. Here, in order to clarify the ambiguous molecular mechanism of IFN-β and evaluate the potential use of them as a biomarker for monitoring of therapy, we investigated the expression of miR-145 and miR-20a-5p in blood sample of 15 treatment naïve RRMS patients, 15 IFN-β-treated RRMS patients, and 15 healthy volunteers (HVs). In silico molecular signaling pathway enrichment analysis was fulfilled on validated and predicted targets of miR-145 and miR-20a-5p to probe the plausible role of them on molecular effects of IFN-β. We identified miR-145 and miR-20a-5p level was normalized in IFN-β-treated patients, and MAPK pathway was one of the most relevant pathways that recognized by molecular signaling pathway enrichment analysis. Moreover, ROC curve analysis of miR-145 indicated that this miRNA could be used for monitoring of response to IFN-β therapy. Restoration of miR-145 and miR-20a expression in IFN-β-treated patients suggests that pleiotropic effects of IFN-β might be through miRNAs. Enrichment of MAPK pathway underscores the importance of non-canonical pathways in IFN-β signaling.