Folate, vitamin B₁₂, and homocysteine (HCY) are involved in the metabolism of nucleic acid precursors and it has been hypothesized that they also influence telomere length, a biomarker of aging. However, previous studies have reported inconsistent findings, and data for older adults are limited. Our study aimed to evaluate associations between leukocyte telomere length (LTL) and serum folate, vitamin B₁₂, and HCY levels among adults aged 55 years and over. In a cross-sectional study in 798 men and women aged 55-79 years, serum folate, vitamin B₁₂, and HCY levels were measured using chemiluminescent immunometric assays, and relative LTL was assessed using quantitative real-time polymerase chain reaction. To evaluate associations between LTL and serum folate, vitamin B₁₂, and HCY levels, multiple linear regression models were used. In multiple models adjusted for age, sex, serum high sensitive C-reactive protein (hs-CRP) levels, and other potential confounding factors, we found no association between LTL and serum folate, vitamin B₁₂, and HCY levels. However, we did find a significant inverse association between HCY levels and LTL in participants with serum hs-CRP levels of ≥ 2 mg/L (p < 0.05). Moreover, there was a trend toward an association between HCY and vitamin B₁₂ levels in these individuals (p = 0.08). In those with serum hs-CRP levels of < 2 mg/L, HCY was inversely associated with vitamin B₁₂ levels (p < 0.001) and had no association with LTL. Our findings suggest that increased serum HCY levels, when combined with the presence of systemic inflammation, may play a role in accelerating biological aging.