Objective: To explore the protocol for diagnosing thyroid nodules based on core needle biopsy (CNB) and study the biomarkers' application in distinguishing indeterminate samples. Methods: Patients with thyroid nodules treated at Peking University First Hospital from 2015 to 2020 were reviewed. In the study, 598 cases with CNB and matched resected specimens were retrieved. According to "diagnostic categories of thyroid CNB" proposed by the Korean Endocrine Pathology Thyroid Core Needle Biopsy Study Group, the CNB samples were diagnosed as follows: Ⅰ, unsatisfactory; Ⅱ, benign; Ⅲ, indeterminate; Ⅳ, follicular neoplasm; Ⅴ, suspicious for malignancy; and Ⅵ, malignant. The samples of CNB Ⅲ were stained by immunohistochemistry (IHC) using antibodies against CK19, Galectin-3, HBME-1, and CD56, and detected by next-generation sequencing (NGS) using an OncoAim^? thyroid cancer multigene assay kit (Singlera Genomics) that detected 26 genes. Taking the resected specimens' classification as the gold standard, the predictive value of CNB for determining the malignancy of thyroid nodules and the biomarkers for distinguishing the samples of CNB Ⅲ was calculated. Results: The study included 598 patients, of which none were CNB Ⅰ, 40 cases were CNB Ⅱ, 40 cases were CNB Ⅲ, 32 cases were CNB Ⅳ, 35 cases were CNB Ⅴ, and 451 cases were CNB Ⅵ. The predictive value of CNB Ⅳ for determining follicular neoplasm was sensitivity (Sen) 100.00% and specificity (Sep) 100.00%, CNB Ⅴ-Ⅵ for determining malignancy was Sen 94.55% and Sep 100.00%, CNB Ⅱ for determining benign lesions was Sen 75.00% and Sep 99.80%. The predictive value of biomarkers for determining malignancy in cases of CNB Ⅲ was Sen 96.30% and Sep 92.31% by NGS, and Sen 81.48% and Sep 92.30% by IHC. Conclusion: The Korean "diagnostic categories of thyroid CNB", which considers the histological specificity of CNB samples and the habits of clinicians, have strong operability, high diagnosis rate, and high clinical value. Under this framework, the cases of CNB Ⅵ should be treated with surgical operation, the cases of CNB Ⅴ-Ⅵ are recommended to be treated as malignant neoplasms, and the major cases of CNB Ⅱ could be followed up without worrisome except the one considered malignant by ultrasound. The value of biomarkers in distinguishing the cases of CNB Ⅲ is significant.