Background The BRAF(V600E) mutation is valuable for the diagnosis, prognosis, and therapy of papillary thyroid cancer (PTC). However, studies related to this mutation have involved only a small number of patients. Therefore, we performed a large-scale analysis from a single institute to evaluate the accuracy of combined fine-needle aspiration (FNA) and BRAF(V600E) mutation tests for PTC diagnosis. Methods A total of 4600 patients with thyroid nodules who underwent both FNA cytology and BRAF(V600E) mutation analysis on FNA specimens were enrolled. The association between the BRAF(V600E) mutation and clinicopathological features was analyzed. A separate analysis was performed for the 311 patients who underwent repeated FNA for comparison of cytological evaluation and BRAF(V600E) mutation results. The diagnostic efficacy of the BRAF(V600E) mutation test and cytologic diagnoses was evaluated for 516 patients who underwent preoperative FNA tests in comparison with conclusive postoperative histopathologic results. Results The cytology results of all 4600 FNA samples were categorized according to The Bethesda System for Reporting Thyroid Cytology (TBSRTC) stages I-VI, which accounted for 11.76%, 60.02%, 6.46%, 3.61%, 6.71%, and 11.43% of the samples, respectively. The BRAF(V600E) mutation was detected in 762 (16.57%) FNA samples, with rates of 1.48%, 0.87%, 20.20%, 3.01%, 66.02%, and 87.81% for TBSRTC I-VI lesions, respectively. Among the 311 repeat FNA cases, 81.0% of the BRAF(V600E)-positive and 4.3% of the BRAF(V600E)-negative specimens with an initial indication of cytological non-malignancy were ultimately diagnosed as malignant by repeat FNA (p < 0.001). Among the 516 patients who underwent thyroidectomy, the sensitivity and specificity of the BRAF(V600E) mutation test alone for PTC diagnosis were 76.71% and 100.0%, respectively, which increased to 96.62% and 88.03%, respectively, when combining the BRAF(V600E) mutation test with cytology. BRAF(V600E) mutation was significantly associated with lymph node metastasis (p < 0.001), but not with age, gender, or tumor size. Conclusions The BRAF(V600E) mutation test in FNA samples has potential to reduce false negatives in PTC diagnosis, and therefore plays an important role in the diagnosis of thyroid nodules, especially those with an indeterminate or nondiagnostic cytology, which should be considered for repeat FNA.