Objectives Graves' disease(GD) is the most common cause of hyperthyroidism. Antithyroid drug(ATD) is the first-line treatment but when discontinued, >50% of patients suffer relapses. Conventional definitive treatment options include surgery and radioiodine therapy(RAI), each with its own disadvantages. Radiofrequency ablation (RFA) achieved promising short-term remission rates in a previous pilot study. The current study reports our experience of using RFA to treat relapsed GD from the largest cohort of patients with longer period of follow-up. Methods This single-arm prospective study recruited consecutive patients aged ≥18 with persistent / relapsed GD requiring ATD from two tertiary endocrine surgery centers. Those with compressive goiter, suspected thyroid malignancy, moderate to severe Graves' ophthalmopathy, preferred surgery/RAI or pregnant were excluded. Eligible patients received ultrasound-guided RFA to the entire bulk of the thyroid gland. ATDs were discontinued afterwards and thyroid function tests were monitored bi-monthly. The primary outcome was disease remission rate at 24-months follow-up after single-session RFA, defined as being biochemically euthyroid or hypothyroid without ATD. Secondary outcomes were complication rates. Results Of the 100 patients considered, 30 (30.0%) patients were eligible and received RFA. Most were female patients (93.3%). The median total thyroid volume was 23mL (15.9 - 34.5). All completed 24-months follow-up. After single-session RFA, disease remission rates were 60.0% at 12-months and 56.7% at 24-months. Amongst the 13 patients with relapse after RFA, 9 (69%) required lower ATD dose than before RFA; 2 received surgery without complications. Total thyroid volume was the only significant factor associated with relapse after RFA (OR 1.054, 95% CI 1.012 - 1.099, p=0.012). At 24-months, RFA led to disease remission in 100% of the 9 patients with total thyroid volume <20ml, and 35% of patients with total thyroid volume ≥20ml (p=0.007). There was no vocal cord palsy, skin burn, hematoma, or thyroid storm after RFA. Conclusions In a highly selected group of patients with relapsed GD and predominantly small thyroid glands, single-session RFA may achieve disease remission. Smaller total thyroid volume may be a favorable factor associated with disease remission after RFA. The results of this study need to be confirmed with a long-term clinical trial.