Objective To investigate the clinical significance of fine needle aspiration biopsy and immunohistochemical markers in differential diagnosis of thyroid nodule.Methods One hundred and eighty-five cases of thyroid nodule were enrolled in the study.All cases received preoperative fine-needle aspiration biopsy and immunocytochemistry.Results The sensitivity,specificity,diagnostic accuracy of fine needle aspiration biopsy to detect diagnosis of thyroid nodules was 75.0％ (21/28),85.4％ (134/157),83.8％(155/185).The positive rates of p53 and galectin-3 immunodetection in discriminating benign from malignant thyroid nodules were 60.7％ (17/28),85.7％ (24/28),combined with p53 and galectin-3 immunodetection in discriminating benign from malignant thyroid nodules was 50.0％ (14/28).The positive rates of p53 and galectin-3 immunodetection in thyroid adenomas were 0,5.1％ (8/157),combined with p53 and galectin-3 immunodetection in thyroid adenomas was 1.3％ (2/157).The positive rates of p53 and galectin-3 immunodetection in malignant thyroid cytologic specimens,alone or combined were significantly higher than those in thyroid adenomas,there were significant difference(P ＜ 0.05).The diagnostic accuracy of fine needle aspiration biopsy with p53 and galectin-3 immunodetection,alone or combined,in discriminating benign from malignant thyroid nodules [94.1％(174/185),93.5％(173/185),91.4％(169/185)] were higher or equal to those in fine needle aspiration biopsy[83.8％(155/185)],there were significant differences (P ＜0.05).Conclusion Immunodetection of p53 and galectin-3 combined with fine needle aspiration biopsy in the differential diagnosis of thyroid nodules can raise the diagnostic accuracy.