<b><i>Introduction:</i></b> Rheumatoid arthritis (RA), a chronic autoimmune disorder, is currently a severe health threat. Previous studies have documented the altered expression of various miRNAs in RA patients. This study determined the expression of miR-124a in RA patients and estimated its diagnostic value for RA. <b><i>Methods:</i></b> A total of 80 RA patients were enrolled as the study subjects, and 36 patients with osteoarthritis were included, with another 36 healthy people as the controls. miR-124a expression levels in peripheral blood plasma, peripheral blood mononuclear cells (PBMCs), and synovial fluid were measured using reverse transcription quantitative polymerase chain reaction, followed by Pearson correlation analysis. Additionally, the association between miR-124a and major clinical indicators was assessed, such as rheumatoid factor (RF), erythrocyte sedimentation rate (ESR), and disease activity score of 28 joints (DAS28). The diagnostic efficacy of miR-124a expression in plasma, PBMCs, and synovial fluid for RA was evaluated by the receiver operating characteristic curve, and the difference in the area under the curve (AUC) was analyzed. <b><i>Results:</i></b> miR-124a was downregulated in RA patients, and the expression levels of miR-124a in plasma, PBMCs, and synovial fluid showed a certain degree of positive correlation. miR-124a was inversely linked with RF, ESR, and DAS28. For the diagnosis of RA patients, the AUC of plasma miR-124a was 0.899 and the cut-off value was 0.800, with 68.75% sensitivity and 94.44% specificity; the AUC of miR-124a in PBMCs was 0.937 and the cut-off value was 0.805, with 82.50% sensitivity and 91.67% specificity; the AUC of miR-124a in plasma combined with PBMCs was 0.961, with a higher diagnostic value than independent plasma or PBMCs; the AUC of miR-124a in synovial fluid was 0.929 and the cut-off value was 0.835, with 80.00% sensitivity and 88.89% specificity. <b><i>Conclusion:</i></b> miR-124a expression is downregulated in the plasma, PBMCs, and synovial fluid of RA patients and has a high diagnostic value for RA.