Abstract Background Hyperhomocysteinemia is associated with autoimmune diseases such as ankylosing spondylitis (AS), systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA). Current findings regarding plasma/serum homocysteine (HCY) levels in AS patients are inconsistent. This study aims to systematically evaluate the association between circulating HCY levels and AS. Methods Online electronic databases (PubMed, Web of Science, Embase, ScienceDirect, China National Knowledge Infrastructure (CNKI), and Wanfang data) were used to retrieve all relevant articles published up to May 7, 2020. The pooled standardized mean difference (SMD) with 95% confidence interval (CI) was calculated using the random-effect model, Stata16 software. Results Nine articles containing 778 AS patients and 522 controls were included in this meta-analysis. No significant differences in HCY levels were found between AS and control groups (pooled SMD = 0.46, 95% CI = − 0.30 to 1.23, P = 0.23). However, subgroup analysis suggested that HCY levels were significantly higher (P < 0.05) in the AS group treated with methotrexate (MTX) compared with the control group. In contrast, HCY levels were significantly (P < 0.05) lower in the AS group receiving anti-TNF-α treatment compared with the control group. No significant differences were detected between HCY levels and disease activity scores (Bath AS disease activity index, BASDAI), and methylenetetrahydrofolate reductase (MTHFR) C677T genotype. Conclusion This meta-analysis indicates that HCY levels are similar between AS and controls, and do not correlate with disease activity. However, different medical treatments cause fluctuations of circulating HCY levels in AS patients. Further and larger-scale studies are needed to confirm these findings. Trial registration This study was registered at international prospective register of systematic reviews (PROSPERO), registration number: CRD42020184426 .