Rheumatoid arthritis (RA) is a persistent autoimmune disorder where serological biomarkers play a crucial role in diagnosis and monitoring disease activity. Antibodies targeting cyclic citrullinated peptides (anti-CCP), mutated citrullinated vimentin (anti-MCV), and rheumatoid factor are commonly used serological markers for RA. However, their respective diagnostic efficacies and potential for mutual complementation remain incompletely understood. This study investigates the diagnostic performance of these three antibodies and their association with disease progression in RA. A total of 257 RA patients who visited Jinhua Hospital Affiliated with Zhejiang University between March and December 2019 were enrolled. Serum specimens were analyzed for anti-CCP, anti-MCV antibodies, and RF levels using chemiluminescence immunoassay (CLIA) and rate nephelometry. The results indicated that the specificity of anti-CCP (94.2%) was higher than that of anti-MCV (84.4%) and RF (84.8%). Furthermore, anti-MCV antibody levels were significantly link to disease duration and morning stiffness. Additionally, anti-MCV and anti-CCP demonstrated differing associations with extra-articular manifestations of RA. The study suggests that anti-MCV antibodies hold significant potential as adjunctive biomarkers in RA, complementing anti-CCP antibodies to improve diagnostic accuracy and provide new insights for early diagnosis and disease monitoring in RA.