Objective To investigate the expression levels of apoptosis-associated speck-like protein （PYCARD）， nuclear factor κB receptor activator ligand （RANKL）， and chitinase 3-like protein 1 （CHI3L1） in the serum of patients with rheumatoid arthritis （RA）， and to explore the relationship between these proteins and disease activity. Methods A total of 104 patients with rheumatoid arthritis （RA group） who were admitted to Anqing Municipal Hospital from January 2021 to December 2025 were recruited for this study. They were divided into the remission group （51 cases） and the active group （53 cases） based on the 28-joint disease activity score （DAS28）. Additionally， 52 healthy volunteers （control group） were recruited during the same period. The expression levels of serum PYCARD， RANKL， and CHI3L1 were compared between the RA group and the control group， as well as between the remission group and the active group. Moreover， the correlation between the expression levels of these three markers and the DAS28 score was analyzed， along with the evaluation value of the combined detection of serum PYCARD， RANKL， and CHI3L1 for the disease activity of RA. Results In the RA group， the expression levels of serum PYCARD， RANKL， and CHI3L1 were significantly higher than those in the control group （P < 0.05）. In the active group， the expression levels of serum PYCARD， RANKL， and CHI3L1 were significantly higher than those in the remission group （P < 0.05）. The expression levels of serum PYCARD， RANKL， and CHI3L1 were positively correlated with the DAS28 score （r = 0.579， 0.534， 0.602， P < 0.05）. The area under the curve （AUC） value of the combined detection of serum PYCARD， RANKL， and CHI3L1 for the evaluation of RA disease activity was 0.898. The AUC values of the three individual tests were 0.743， 0.730， and 0.767， respectively. The AUC value of the combined detection was significantly higher than those of the individual detections （P < 0.05）. The sensitivity and specificity of the combined detection were 92.45% and 84.31%， respectively. Conclusions The expression levels of PYCARD， RANKL， and CHI3L1 are related to the onset of RA and its disease activity. The combined detection of PYCARD， RANKL， and CHI3L1 has the advantage of collaborative diagnosis and is of high value in evaluating the disease activity of RA， providing a more objective and accurate assessment method for the disease activity status of clinical RA patients.