Objective To investigate the differences of the level of homocysteine (Hcy) between ALS patients and controls. Methods PubMed, EMBASE, OVID, and other databases were searched systematically up to October 2022 for relevant reports about the level of Hcy, folic acid, and vitamin B12 (VB12) among ALS patients. Two reviewers screened and selected the titles and abstracts of the studies independently during the database searches and performed full-text reviews and extracted available data. The MD (mean difference) and 95%CI (credibility interval) of the level of Hcy, folic acid, and VB12 between ALS group and control group were calculated. Results Pooled results of nine studies including 812 ALS patients and 2632 controls showed that the MD in plasma levels of HCY between ALS patients and controls was 1.56 (95%CI: - 0.07, 3.19) mu mol/L with remarkable heterogeneity (I-2 = 94%). The mean CSF levels of Hcy among ALS patients were significantly higher than that of controls (MD: 0.23, 95%CI: 0.21, 0.24 mu mol/L) with no significant heterogeneity (I-2 = 0%). No significant difference in the plasma level of folic acid (MD: - 0.52, 95%CI: - 1.89, 0.84 ng/mL) or VB12 (MD: - 9.76, 95%CI: - 83.41, 63.89) was found between ALS patients and controls. Conclusion There was no significant difference in the plasma level of Hcy, folic acid, or VB12 between ALS patients and controls. The CSF level of Hcy among ALS population was remarkably higher than that among controls. Vitamin supplements including folate and VB12 might be recommended to ALS patients with the complication of deficiencies.