To investigate the effect of ultrasound combined with expression of Galectin-3, c-Met, HBME-1 and CK19 in differentiating malignant from benign thyroid nodules. Forty-six patients with thyroid nodules were studied with ultrasound and immunohistochemical staining of excised thyroid nodules. The data were classified and compared. The immunohistochemical staining revealed 8 benign and 41 malignant thyroid lesions. In ultrasound risk assessment, the malignancy risk was low in four nodules, medium in five and high in 37 with lymphatic metastasis in 26. A significant (P<0.05) association existed in the expression of Galectin-3 with nodule boundary and lymphatic metastasis, in HBME-1 with nodule micro-calcification and in c-Met with nodule micro-calcification and lymphatic metastasis. CK19 expression was not significantly (P>0.05) associated with any of ultrasound features of nodule. Galectin-3, c-Met, HBME-1 and CK19 were significantly (P<0.05) different in malignant and benign thyroid lesions, with a significant (P<0.01) tendency in all the molecular markers in predicting the malignant from benign lesions. The ultrasound characteristics could significantly (P<0.001) predict malignant nodules with a significant (P<0.05) prediction tendency. The scores of Galectin-3, c-Met and CK19 significantly (P<0.05) increased with increase of ultrasound malignancy risk degree. In malignant and benign lesions differentiated by ultrasound, no significant (P>0.05) difference existed in HBME-1 expression, however, with ultrasound malignancy risk increase, the score of HBME-1 expression increased significantly (P=0.03). Galectin-3, c-Met, HBME-1 and CK19 have significantly greater expressions in thyroid malignant than benign lesions and their expression increases with increase of ultrasound malignancy risk. The combination of both ultrasound and molecular markers can be used to differentiate malignant and benign thyroid lesions.