Objective To evaluate the clinical significance of preoperative serum thyroglobulin (Tg), neck ultrasound and 99TcmO4- thyroid static imaging in identification of benign and malignant thyroid nodule. Methods 103cases of thyroid nodules with the surgical and pathological confirmed were retrospectively carried out data analysis. According to preoperative examination, the patients were divided into 3 groups: 31 cases with three full examination (Group A), 43 cases with the neck ultrasound and 99TcmO4- thyroid static imaging (Group B), and 29 cases with the neck ultrasound (Group C). The sensitivity, specificity and accuracy of each examination were obtained based on the postoperative pathological results. Results The sensitivity in Group A, B and C were 28.5%, 83.3%, and 56.2%; the specificity were 58.3%, 58.1% and 46.2%; and the accuracy were 51.6%, 65.1%, and 51.7% , respectively. The sensitivity, specificity and accuracy by combined neck ultrasound and 99TcmO4- thyroid static imaging (Group B) were significantly higher than the single neck ultrasound inspection (Group C). There were no significant advantages in Group A over Group B and C. Conclusion The preoperative neck ultrasound and 99TcmO4- thyroid static imaging examination for differential diagnosis of thyroid nodules would be necessary.