Objective To establish and perfect the serological test system for diagnosis of rheumatoid arthritis (RA), search for new autoantibody markers of RA, and explore the role and clinical significance of these parameters in occurrence and development of RA. Methods Human fibrinogen (hFib) and bovine type Ⅱ collagen (bCⅡ) were modified and citrullinated by peptidylarginine deiminase (PAD), respectively. CⅡ peptide (CⅡ 260272) and cyclic citrullinated peptide (CCP) were selected as immobilized antigens. The enzymelinked immunosorbent assay (ELISA) for autoantibodies against Cit-hFib, Cit- bCⅡ, CⅡ peptide and CCP antigens were established. The positive rates of these autoantibodies in clinical patients and the relationship with other laboratory parameters were also analyzed. The diagnostic value of these autoantibodies was assessed in the patients with RA. Results The positive rates of antiCit-hFib and antiCit-bCⅡ antibody in the patients with RA were 59.4% and 56.3% respectively, and were significantly higher than those in the patients with other diseases and healthy controls (P<0.01). The sensitivity and specificity of these antibodies for RA diagnosis were significantly higher than those of anti hFib and antibCⅡ antibody. The diagnostic value of antiCⅡ peptide antibody was close to that of antiCCP antibody. The diagnostic sensitivities of antiCⅡ peptide and antiCCP antibody for RA were of 67.2% and 68.8%, and the specificities were of 95.3% and 97.8%. The positive/negative predictive values were of 76.8%/88.0% and 92.6% /93.1% respectively. The positive/negative likelihood ratios were 14.3/31.3 and 0.34/0.32 respectively. Youden indexes were up to 0.625 and 0.666. In 13 patients with early stage of RA, the frequencies of both antiCⅡ peptide and antiCCP antibody were up to 76.9%. The area under the ROC curve of antiCCP antibody was of 0.946, and antiCⅡ peptide antibody was of 0.924. The combination of antiCⅡ peptide and antiCCP antibody or antiCⅡ peptide and rheumatoid factor could effectively improve the diagnostic level of RA. The results of correlation analysis and symmetric measurement showed that each autoantibody was significantly correlated with antiCCP antibody. Among them, antiCit-hFib and antiCit-bCⅡ antibodies presented synchrony with antiCCP antibody to a certain extent (0.4