Objective To explore the serum homocysteine (Hcy) levels in patients with rheumatoid arthritis (RA) and analyze the relationship between Hcy and RA disease activity as well as treatment response. Methods A total of 58 patients with newly diagnosed rheumatoid arthritis (RA) admitted to the Department of Rheumatology and Immunology of Jining NO.1 People's Hospital from August 2022 to December 2023 were enrolled. 58 healthy subjects undergoing physical examination at the Physical Examination Center of our hospital during the same period were selected as the control group. The correlation between Hcy levels and laboratory indicators of RA as well as DAS28 (ESR) score in the RA group was analyzed. According to the DAS28 (ESR) score, the RA patients were divided into two subgroups: DAS28≥3.2 group and DAS28＜3.2 group. The Hcy levels and laboratory indicators were compared between the two subgroups and the healthy control group. The RA patients were treated with antirheumatic drugs for 3 months, and the Hcy levels and DAS28 (ESR) scores before and after treatment were compared. Results The Hcy level in RA group was higher than that in healthy control group (P＜0.05). The level of Hcy in the group with DAS28≥3.2 was higher than that in the group with DAS28＜3.2 and the healthy control group (P＜0.05), while there was no significant difference in Hcy level between the group with DAS28≥3.2 and the healthy control group (P＞0.05). The levels of rheumatoid factor(RF), ESR, C-reactive protein(CRP), and IL-6 in the group with DAS28≥3.2 were higher than those in the group with DAS28＜3.2 (P＜0.05), but there was no significant difference in anti-cyclic citrullinated peptide antibody(anti-CCP), tumor necrosis factor-α(TNF-α) level between the two groups (P＞0.05). Serum Hcy levels exhibited a positive correlation with ESR, IL-6 levels, and DAS28 (ESR) in the RA patient group. However, no significant correlation was observed between Hcy and RF, anti-CCP, CRP, TNF-α. A fter treatment with antirheumatic drugs, Hcy level in patients with RA was lower than that before treatment (P＜0.05) and the DAS28 (ESR) score after treatment was lower than that before treatment (P＜0.05).Conclusion In patients with newly diagnosed RA, elevated Hcy levels are positively correlated with indicators of disease activity. After treatment with antirheumatic drugs, the disease activity of patients decreases and Hcy levels also decline accordingly. This suggests that Hcy may be involved in the pathogenesis of RA and is expected to serve as a new indicator for evaluating RA disease activity