Objective To investigate the clinical value of contrast-enhanced ultrasound (CEUS) in differential diagnosis between benign and malignant gallbladder lesions which diameter more than 1 cm. Methods A retrospective analysis included CEUS data of 142 cases with gallbladder lesions diameter more than 1 cm. All lesions were confirmed by surgical pathology, enhanced CT/MRI or clinical diagnosis with following up more than 2 years. To summarize the CEUS characteristics of benign and malignant gallbladder lesions over 1 cm in diameter, the CEUS characteristics of benign and malignant gallbladder lesions diameter more than 1 cm were analyzed, referring to the gallbladder CEUS guidelines. The contrast medium washout before 35 s and 60 s were used as the combined diagnosis with CEUS standard to identify gallbladder cancer, and the diagnostic efficiency was calculated respectively. Results There were statistical differences between the malignant group and the benign group in terms of CEUS enhancement pattern, contrast arrival time, washout time, and gallbladder wall continuity, as well as enhancement levels (P<0.001). The sensitivity, specificity, accuracy in the diagnosis of gallbladder malignant lesions were 93.0%, 83.8%, and 86.6% based on CEUS standard. The sensitivity, specificity and accuracy of diagnosis gallbladder malignant lesions were increased to 93.0%, 92.9% and 93.0% when combined with washout before 35 s of contrast medium. Conclusions CEUS is highly valuable in differentiating benign and malignant gallbladder lesions diameter more than 1 cm. Combined with contrast medium washout before 35 s is helpful to improve the ability of differentiating benign and malignant gallbladder lesions.