Abstract: Objective To investigate the expression of miR-326 and miR-223 in peripheral blood mononuclear cells (PBMC) and plasma in patients with rheumatoid arthritis (RA) and their clinical significances. Methods One hundred and fifty-two patients with RA, 65 patients with Osteoarthritis (OA) and 65 healthy controls in Xiangya Hospital and People’s Hospital of Danzhou from January 2015 to June 2017 were enrolled in this study. PBMC and plasma miR-326 and miR-223 expression levels were detected by RT-PCR. The diagnostic value of PBMC and plasma miR-326, miR-223 and rheumatoid factor (RF) in RA were evaluated by ROC curve. Pearson correlation analysis was used to explore the correlation between PBMC and plasma miR-326, miR-223, RF, C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) in RA patients. Results The expression of PBMC/ plasma miR-326 and miR-223 in the RA group were significantly higher than those in the OA group and the control group PBMC/plasma miR-326 (2^-ΔΔCt): (0.12±0.05) vs (0.05±0.03) and (0.04±0.03), (3.56±1.04) vs (1.24±0.36) and (1.13±0.32); PBMC/plasma miR-223 (2^-ΔΔCt): (0.42±0.13) vs (0.21±0.08) and (0.18±0.06), (6.95±2.16) vs (5.13±1.42) and (4.86±1.25), all P< 0.05. The expression of PBMC/ plasma miR-326 and miR-223 in the high activity group were significantly higher than those in the low active group PBMC/plasma miR-326(2^-ΔΔCt): (0.18±0.09) vs (0.05±0.04), (5.62±1.58) vs (1.43±0.52); PBMC/plasma miR-223(2^-ΔΔCt): (0.67±0.24) vs (0.23±0.12), (8.28±2.63) vs (5.47±1.48), all P< 0.01. The AUC of miR-326, miR-223, RF and the combination of three markers in diagnosis of RA were 0.805(0.745-0.867), 0.824(0.761-0.893), 0.762(0.697-0.828), 0.893(0.825-0.961) for PBMC, and 0.882(0.817-0.953), 0.837(0.775-0.906), 0.852(0.786-0.918), 0.936(0.868-0.996) for plasma, respectively.Correlation analysis showed that in RA group, PBMC/plasma miR-326 was positively correlated with RF, miR-223 and CRP (AllP< 0.05), plasma miR-326 and miR-223 were positively correlated with ESR (P< 0.05), plasma/PBMC miR-326 was positively correlated with miR-223 (P< 0.01). Conclusion miR-326 and miR-223 are abnormally high in PBMC and plasma in patients with RA, and they are related to the activity of RA, which can be used as a biological marker for RA diagnosis.