Objectives: To evaluate the expression and significance of CK19, TPO, and HBME-1 in the differential diagnosis of papillary thyroid carcinoma (PTC) and nonmalignant nodules. Methods: Tissue samples were obtained from 257 patients with PTC and 149 patients with nonmalignant thyroid specimens, and immunohistochemical staining for CK-19, TPO, and HBME-1 was performed. Results: The expression of CK-19, TPO, and HBME-1 was 96.3%, 12.0%, and 85.3%, respectively, for the PTC group. For nonmalignant thyroid lesions group, the expression of these markers was 40.4%, 86.2%, and 37.2%, respectively. Further, the expression of CK-19 and HBME-1 in PTCs was much higher than that in the benign thyroid lesions (P < 0.05). However, the positive expression of TPO in PTC specimens was much lower than that in the nonmalignant specimens (P < 0.05). CK-1 had the highest sensitivity (96.30%) for PTCs. The combination of the positive expression of CK-19 and negative expression of TPO had the highest sensitivity (98.50%), while that of the positive expression of HBME-1 and negative expression of TPO had the highest specificity (92.90%). Conclusions: The combination of positive expression of CK-19 or HBME-1 or negative expression of TPO can improve the specificity of the diagnosis of PTC.