Multiple sclerosis (MS) is an autoimmune neurodegenerative disorder, with relapsing-remitting MS (RRMS) being the most common subtype. Interferon-γ (IFN-γ) plays a dual role in MS pathogenesis. MicroRNAs (miRNAs) have emerged as potential diagnostic biomarkers. This study examined the effect of relative expression of hsa-miR-24-3p and hsa-miR-181d-3p, plasma IFN-γ levels, and the IFNG rs2069727 T/C variant on MS risk, evaluating their interrelationships and diagnostic potential. This case-control study comprised two overlapping groups-a genetic polymorphism group (330 RRMS, 330 healthy controls (HCs)) and a miRNA group (25 glatiramer acetate (GA)-treated RRMS patients, 25 treatment-naïve RRMS patients, and 25 HCs)- collected at the Ankara Bilkent City Hospital Neurology Polyclinic. The IFNG rs2069727 T/C variant did not display a statistically significant disparity between RRMS patients and HCs. Significantly elevated hsa-miR-24-3p and hsa-miR-181d-3p relative expression levels were observed in GA-treated and treatment-naïve RRMS patients compared to HCs. Conversely, age-adjusted plasma IFN-γ concentrations were markedly lower in GA-treated and treatment-naïve RRMS patients versus HCs. Individuals with low plasma IFN-γ levels (≤ 1.311 pg/mL) demonstrated significantly elevated hsa-miR-24-3p relative expression compared to the high IFN-γ group (> 1.311 pg/mL). Conversely, subjects with low hsa-miR-181d-3p levels (≤ 2.90) exhibited significantly higher plasma IFN-γ concentrations relative to those with high hsa-miR-181d-3p levels (> 2.90). In the GA-treated group, EDSS negatively correlated with age-adjusted plasma IFN-γ. This study identified age-adjusted plasma IFN-γ, hsa-miR-24-3p, and hsa-miR-181d-3p expression as potential blood-based biomarkers for RRMS diagnosis and analyzed them alongside disability scores. The miRNAs in this study can be further evaluated as prospective therapeutic targets.