Objective To study the clinical value of single cardiac cycle real-time three dimensional transthoracic echocardiography (RT3DE) in the diagnosis and treatment of atrial septal defect in children. Methods GE Vivid E9 ultrasound system and a M5S wide-angle or a 4V-D (4D cardiac) probe were applied in 27 patients with congenital atrial septal defect (ASD) to perform conventional two-dimensional transthoracic echocardiography (2DE) and single cardiac cycle RT3DE and to make the diagnosis and assess the size and location of the defects. The findings of 2D and RT3D echocardiography were compared with surgical results. Results Single cardiac cycle RT3DE stereoscopically displayed atrial septum simulating surgical view from multiple perspectives, including overviews of the shape, position, size, and proximity of defects. ASD maximal diameters measured by RD3DE (29.7±7.3) mm had better correlations (r=0.96, r=0.82, respectively) with surgical measurements(31.2±9.2) mm than by 2DE(33.6±9.2) mm. Conclusions Single cycle RT3DE can simulate surgery views and provide a new approach for accurate and reliable the measurement of ASD, so it has important potential for clinical applications.