Background and Aims Thyroid nodules are common clinical entities. Among them, malignant nodules have a higher risk of metastasis. Correct definition of the nature of thyroid nodules is of great significance for developing treatment plans and improving prognosis. However, all currently available inspection methods have their limitations. Therefore, this study was performed to investigate the efficiency of spectral CT and its combination with multiple other factors in differential diagnosis of thyroid nodules.Methods The data of patients with thyroid nodules admitted from August 2018 to March 2021 were reviewed, and the eligible cases were selected according to the inclusion and exclusion criteria. The demography factors, imaging findings, spectral data and thyroid function test results of between patients with benign or malignant thyroid nodules were analyzed, and the variables with significant difference were screened out. The receiver operating characteristic (ROC) curve was draw, and the diagnostic efficiency was evaluated by the value of the area under curve (AUC).Results A total of 151 patients were enrolled, of whom 46 were males and 105 were females with a total of 224 nodules that included 61 benign nodules and 163 malignant nodules. The variables showing significant difference between patients with benign or malignant thyroid nodules were age (P<0.001), thyroglobulin (Tg) (P<0.001), thyroid peroxidase antibody (P=0.003) and calcitonin (P=0.017); the variables with significant difference in terms of morphological features and spectral parameters between benign and malignant thyroid nodules included the boundary (P<0.001), calcification (P=0.03), cystic degeneration (P<0.001), and rate of change of water concentration (RC_WC) (P=0.002). The results of multivariate analysis revealed that absent of cystic degeneration, RC_WC, Tg and age were risk factors for malignant nodules (all P<0.05). The sensitivity, specificity, accuracy and AUC for spectral CT alone (spectral parameters plus imaging characteristics) were 55.7%, 94.4%, 91.0% and 0.783, for spectral CT combined with Tg detection were 81.0%, 80.9%, 80.9% and 0.850, and for spectral CT combined with Tg and age were 85.7%, 84.6%, 84.7% and 0.905, respectively.Conclusion Spectral CT has certain value in differential diagnosis of benign and malignant thyroid nodules. The RC_WC and imaging characteristics obtained from spectral CT combined with Tg detection, with consideration of age of the patients, may offers a higher accuracy for definition of the nature of thyroid nodules.