Objective To analyze the serum levels of matrix metalloproteinase-3 (MMP-3), glucose-6-phosphate isomerase (GPI) and anti-cyclic citrulline peptide containing citrulline (anti-CCP) markers in patients with rheumatoid arthritis (RA), and explore their clinical value in the diagnosis of rheumatoid arthritis (RA). Methods 101 patients with rheumatoid arthritis who were treated in the Department of Rheumatology and Immunology of our hospital from March 2022 to November 2022 were selected as the experimental group. 96 healthy people were selected as the control group. MMP-3, GPI and anti-CCP were detected by enzyme-linked immunosorbent assay (ELISA), RF, ASO, CRP, C3, C4 were detected by immunoturbidimetry, ESR was detected by automatic ESR, and 25(OH)D3 was detected by electrochemiluminescence method. The statistical differences between MMP-3, GPI and anti-CCP, RF, ASO, CRP, C3, C4, ESR and 25(OH)D3 in the serum of the two groups were analyzed, ROC curve to analyze the diagnostic value of single test and combined test for RA. Results The serum levels of MMP-3, GPI, anti-CCP, RF, CRP, C3, C4, ESR in patients with rheumatoid arthritis were significantly higher than those in the control group (P＜0.05). The ASO, 25 (OH) D3 were significantly lower than those in the control group (P＜0.05). The ROC curve showed that the maximum area of MMP-3+GPI+anti-CCP curve was 0.997, the sensitivity was 97.8%, the specificity was 94.7%, and MMP-3, GPI Anti-CCP was an independent risk factor for RA. Conclusion The combined detection of MMP-3, GPI and Anti-CCP has high diagnostic efficacy and can provide evidence for the diagnosis of RA