BACKGROUND Although fluoroless transseptal puncture (TSP) guided by intracardiac echocardiography (ICE) has been used for many years, there are no reports of an accurate site-specific method for TSP in detail, especially about the safety and efficiency of the method. This study aimed to compare the efficacy and safety of TSP guided by three-dimensional ICE using a fluoroless site-specific method with that of the conventional fluoroless method in patients with atrial fibrillation (AF). METHODS This prospective study included 60 patients with AF scheduled for radiofrequency ablation who were assigned to undergo modified fluoroless site-specific TSP (SS-ICE group, n = 30) or conventional fluoroless TSP (C-ICE group, n = 30). TSP was guided by three-dimensional ICE in both study groups. RESULTS All fluoroless TSP were performed successfully in both groups. There were no significant differences in patient characteristics, Pre-TSP time (11.3 ± 1.7 min vs. 11.1 ± 1.6 min, P = 0.822) and TSP time (3.4 ± 0.9 min vs. 3.5 ± 1.1 min, P = 0.772) between the SS-ICE group and the C-ICE group. The distance between the actual traversing point and the presetting point in the fossa ovalis was less than 5 mm in 87% of patients (26/30, 3.1 ± 1.2 mm) in the SS-ICE group. There were no TSP-related complications in either group. CONCLUSIONS SS-ICE method is a simple, safe, and effective approach for fluoroless site-specific TSP.