Background: microRNAs (miRNAs) regulate the expression of the genome at the post-transcriptional level. They play a role in autoimmunity and inflammation, and show potential for use as therapeutic targets in many diseases. With the recent detection of miRNAs in body fluids, the possibility for using miRNAs as diagnostic biomarkers has emerged. Objective: We assessed whether miRNAs contribute to the altered immune activation state in relapsing–remitting multiple sclerosis (RRMS) patients and investigated the possible use of miRNAs as diagnostic biomarkers in multiple sclerosis (MS). Methods: We performed global miRNA expression profiling analysis in peripheral blood mononuclear cells (PBMCs) and selected miRNAs were measured in plasma. We detected expression of miRNAs by real-time qPCR and compared results with cytokines related to inflammation and disease activity. Selected miRNAs were analyzed in PBMC subpopulations, after isolating them by magnetic bead separation. Results: We found that among validated miRNAs, let-7d correlated with the pro-inflammatory cytokine interleukin-1B. The miR-145 was 3-fold up-regulated in MS patients; its possible use as a diagnostic biomarker in PBMCs, plasma and serum was confirmed by ROC-curve analysis (Area under the curve (AUC) 0.785, p = 0.0004; 0.785, p = 0.004; 0.981, P < 0.0001, respectively). Conclusions: RRMS patients in remission had altered expression of miRNAs. We validated miR-145 as a potential diagnostic biomarker for the diagnosis of MS in blood, plasma and serum.