Background Elevated homocysteine (Hcy) has been reported to be associated with cardiovascular events in atrial fibrillation (AF) patients, while the age-related expression pattern of plasma Hcy in AF remains unknown. The study was aimed to investigate the effect of advanced age on plasma Hcy levels and its association with ischemic stroke in non-valvular AF patients. Methods A total of 2562 consecutive patients with non-valvular AF and 535 controls were enrolled and divided into six age groups. Plasma Hcy levels were analyzed among different age groups, and the effect of advanced age on Hcy was investigated. Results Plasma Hcy levels did not show any difference among groups aged below 65 years, while it increased sharply in patients aged 65-74 years and aged over 75 years (15.7 +/- 4.6 mu mol/L, 17.1 +/- 4.9 mu mol/L, both P < 0.01 compared with the first four age groups). Hcy was much higher in AF patients than in controls at the same age group (all P < 0.05). The proportion of patients with hyperhomocysteinemia increased gradually with age from 32.3%, 29.2%, 31.2%, 32.4%, 45.9%, to 51.4% in six age groups. The concentration of Hcy in AF patients with ischemic stroke increased progressively with age, and was higher than those without stroke at the same age. Logistic regression analysis demonstrated that age 65-74 years [odds ratios (OR): 1.742, 95% confidence interval (CI): 1.223-2.482, P = 0.002] and age >= 75 years (OR: 2.637, 95% CI: 1.605-4.335, P < 0.001) were significantly independent predictors of elevated plasma Hcy levels. Conclusions Advanced age was significantly associated with elevated Hcy levels, which may provide a possible explanation for the progressive increase in ischemic stroke especially in elderly AF patients.