The aim of the study was to examine changes in left atrial appendage volume (LAA-V) in patients with non-valvular atrial fibrillation (AF) using real-time three-dimensional transesophageal echocardiography (RT3D-TEE) and evaluate the prediction value on the high risk of thrombosis of LAA. Using RT3D-TEE we measured: i) LAA peak empty velocity (LAA-PEV), ii) LAA-V including LAA end-diastolic volume (LAA-EDV) and end-systolic volume (LAA-ESV). We also calculated LAA ejection fraction (LAA-EF). RT3D-TEE was applied in 20 control cases and 74 patients with non-valvular AF. According to the presence of thrombosis, 74 patients were divided into the no thrombosis group (NTH group, n=52) and thrombosis group (TH group, n=22). Our results showed that there were significant differences in LAA-V and LAA-EF values in different groups (P<0.05). LAA-EDV moderately correlated with LAA-PEV (r=−0.531, P<0.001) while LAA-ESV demonstrated a strong correlation with LAA-PEV (r=−0.741, P<0.001). LAA-EF also showed a strong correlation with LAA-PEV (r=0.693, P<0.001). Through receiver operating characteristic (ROC) curves, the cut-off values of LAA-EDV and LAA-ESV in thrombosis of LAA were 18.45 and 9.69 ml, respectively. RT3D-TEE effectively evaluated the LAA-V, LAA-PEV and LAA-EF parameters, and proved to be valuable in the process of evaluation of thrombosis of LAA.