Objective To investigate the value of double contrast-enhanced ultrasonography (DCEUS) in combination with three-dimension (3D) contrast-enhanced ultrasound in diagnosis of benign or malignant gastric ulceration. Methods A total of 47 patients with gastric lesions were enrolled in this study. All have the pathological results, 22 of them were benign ulceration and 25 were malignant. All patients underwent DCEUS and 3D contrast-enhanced ultrasound. On the basis of oral contrast-enhanced ultrasound agents, intravenous contrast-enhanced ultrasound agents were given at the same time. Images and data were recorded. The arrival time (AT), time to peak (TTP), infusion time (IT), baseline intensity (BI), peak intensity (PI) and enhanced intensity (EI) were calculated using the time-intensity curve (TIC). Results All benign ulcerations were imaged as small pits with slight thicken of gastric wall [(8.66±2.87) mm] using DCEUS,while malignant lesions were relatively large irregular-shape, unevenly-bottom pits with significant thicken of gastric wall [(13.98±3.63)~(20.83±3.69) mm]. 3D-DCEUS images were analyzed for gastric mucosal folds, ulceration lesions, as well as local vascularity. All ulceration lesions showed broken of gastric mucosal folds, and malignant lesion showed thickness of gastric wall as“crater”, with rich and irregular vascularity. There were no statistical differences between benign gastric ulcerations and adjacent normal gastric wall (P>0.05). Malignant gastric ulceration were compared with adjacent normal gastric wall tissue, TTP, IT and BI had no statistical differences (P>0.05), while AT, PI, and EI had statistical differences [(9.00±2.02) s vs (10.90±2.75) s, P=0.008], [(35.46±5.77) dB vs (29.73±8.72) dB, P=0.009] and [(30.76±5.76) dB vs (23.45±6.84) dB, P=0.000]. PI and EI in malignant tumors were higher than those in benign ones (P<0.05). Conclusion DCEUS could be a new method in differentiating benign and malignant gastric ulceration, which can get both the anatomy and perfusion information of gastric wall as well as lesions. 3D-DCEUS can improve the quality of the conventional ultrasound image.