Objective To investigate the application value of Real-time three-dimensional Transesophageal echocardiography (RT-3D-TEE) in percutaneous closure of patent foramen ovale (PFO). Methods The case data and follow-up data of 95 patients with percutaneous PFO occlusion under the guidance of RT-3D-TEE were retrospectively analyzed, and to analyze the application value of RT-3D-TEE in improving the success rate of surgery, shortening the operation time and ensuring the safety of surgery. Results RT-3D-TEE could show the 3D-anatomy structure of heart and occlusion device on line. It could detect the position, size and shape of PFO, guiding and monitoring percutaneous PFO occlusion. Among the 95 patients, 94 cases were successfully occlusion, the success rate was 98.95%, the mean operation time was (26.81±5.98) min, transient arrhythmia occurred in 4 cases (4.21%), and no serious complications occurred during 1-3 years of postoperative follow-up. Conclusion RT-3D-TEE could show the 3D-anatomy structure of heart on line for percutaneous PFO occlusion, which is conducive to the communication between echocardiographers and surgeons, and provides a guarantee for the high success rate, short operation time and safe completion of the PFO occlusion operation. Therefore, it has important application value.