Objective To investigate the clinical application of cytokeratin 19 (CK19), galectin-3 (Gal-3), p53, topoisomerase Ⅱ (TopoⅡ), Ki-67, cyclin D1 (CCND1), human bone marrow endothelial marker 1 (HBME-1), and B-Raf proto-oncogene, serine/threonine kinase V600E mutant type (BRAF^V600E) in the pathological diagnosis of papillary thyroid carcinoma (PTC). Methods A total of 100 PTC and 40 benign thyroid lesions were selected. The expression of CK19, Gal-3, p53, TopoⅡ, Ki-67, CCND1 and HBME-1 in tissues was detected by immunohistochemistry EnVision method, and the expression of BRAF^V600E was detected by fluorescence polymerase chain reaction. Results The positive expression rates of CK19, Gal-3, TopoⅡ, CCND1, HBME-1 and BRAF^V600E in PTC tissues (97.0%[97/100], 98.0%[98/100], 56.0%[56/100], 95.0%[95/100], 66.0%[66/100] and 75.0%[75/100]) were significantly higher than those in benign thyroid tissues (45.0%[18/40], 20.0%[8/40], 15.0%[6/40], 55.0%[22/40], 25.0%[10/40] and 12.5%[5/40]), and the differences were statistically significant (all P<0.01). CK19 and Gal-3 had the highest sensitivity (97.0% and 98.0%, respectively) and Gal-3 had the highest accuracy (92.9%) when the 8 markers were used alone for the diagnosis of PTC. Taking any 2 being positive of a 3 combination as the positive criterion, the combined application of CK19, Gal-3 and HBME-1 as diagnostic criteria for PTC had the highest accuracy of 96.4%. The combication of BRAF^V600E, CK19 and Gal-3 showed high accuracy as well (91.4%). Conclusion CK19, Gal-3, TopoⅡ, CCND1, HBME-1 and BRAF^V600E are important markers for the diagnosis of PTC, and the combined application of these markers can help to distinguish the abnormal PTC from benign thyroid lesions.