It has been reported that citrullinated fibrin(ogen) deposits in the inflamed joints played an important role in the pathogenesis of rheumatoid arthritis (RA). Although antibodies to citrullinated fibrinogen (ACF) have been detected in the sera of RA patients, the associations between ACF and RA remain unclear. In this study, human fibrinogen was citrullinated by peptidylarginine deiminase in vitro, and the ACF were detected by an enzyme-linked immunosorbent assay in rheumatic patients, including 183 RA, 121 systemic lupus erythematosus, 48 osteoarthritis, and 108 healthy controls. The prevalence of ACF was determined, and the associations between ACF and RA were evaluated. It was shown that the sensitivity and specificity of ACF in RA were 67.21 and 84.84%, respectively. There were significant correlations between ACF and erythrocyte sedimentation rate, anti-cyclic citrullinated peptide antibody, and anti-keratin antibodies (AKA). In radiographic progression, the RA patients with ACF had higher scores than those without ACF according to the Sharp-van der Heijde method. In addition, ACF was often positive in the RA patients who were IgM rheumatoid factor negative or AKA negative or anti-perinuclear factor negative. The results indicate that ACF assay is helpful for the diagnosis of RA.