Rheumatoid arthritis (RA) is one of the most common systemic autoimmune diseases. New markers are needed for early diagnosis of RA as seronegativity in both early and established RA remains a major limitation of both anticitrullinated protein antibodies (ACPA) and rheumatoid factor (RF). The 14-3-3η protein may represent a novel biomarker for the detection of RA. We evaluated the diagnostic performance of serum 14-3-3η protein in early and established cases of rheumatoid arthritis and we compared the diagnostic accuracy of it with those of the well-known RA markers (e.g. RF and ACPA). Sera from 50 patients with RA (20 early and 30 established) based on the 2010 ACR / EULAR Rheumatoid Arthritis Classification Criteria, 15 patients with non-RA arthritis as diseases control group (8 patients with OA and 7 patients with SLE) and 14 healthy controls were enrolled in the study. Serum RF was determined by latex, ACPA and 14-3-3η protein were determined by ELISA. Serum 14-3-3η protein levels in patients with RA were significantly higher (P=0.001*) as compared to healthy individuals. For serum 14-3-3η diagnostic accuracy in RA; Receiver operating characteristic curves (ROC) analysis comparing patient with RA with healthy control showed AUC (0.916) at optimum cutoff of > 2.5ng/mL, and a sensitivity of 100%, a specificity of 78.57%, a PPV of 94.3, and an NPV of 100. No significant difference in 14-3-3η protein serum levels was found between early and established RA groups. It was positive in 100% of early and established RA patients who were seronegative for RF and ACPA. It is concluded that, 14-3-3η protein could improve the sensitivity of RA diagnosis and cover the shortage of detection of RF and ACPA in RA patients.