Background: Papillary thyroid cancer cells can express oestrogen receptor alpha, which is encoded by the ESR1 gene and may bind to oestrogen to induce the occurrence and development of papillary thyroid cancer. The BRAF(V600E) mutation is also an important biomarker for the occurrence and progression of papillary thyroid cancer. However, the association between the BRAF(V600E) mutation and oestrogen receptor alpha expression has not been identified. This study aims to investigate the association between ESR1 expression and the BRAF(V600E) mutation and its clinical significance. Methods: Oestrogen receptor alpha and BRAF(V600E) proteins were detected by immunohistochemical staining of formalin-fixed paraffin-embedded thyroid tissues from 1105 patients with papillary thyroid cancer at our institution. Messenger RNA expression counts of ESR1 and clinicopathologic information were obtained from The Cancer Genome Atlas database. Results: Oestrogen receptor alpha protein expression was significantly associated with BRAF(V600E) protein. The positive rate of oestrogen receptor alpha protein in papillary thyroid cancer patients was significantly higher in males, younger patients and patients with the multifocal type. In papillary thyroid cancer patients with positive BRAF(V600E) protein, oestrogen receptor alpha expression was significantly correlated with central lymph node metastasis. Data from the The Cancer Genome Atlas database also suggested that the ESR1 messenger RNA level was associated with the BRAF(V600E) mutation. Furthermore, classification analysis performed according to a tree-based classification method demonstrated that higher ESR1 messenger RNA expression indicated poorer overall survival in papillary thyroid cancer patients with the BRAF(V600E) mutation. Conclusions: The percentage of BRAF(V600E) mutations is increased in patients with higher ESR1 messenger RNA levels, and the BRAF(V600E) protein might be co-expressed with oestrogen receptor alpha, which could be an indicator of cervical lymph node metastasis and poor overall survival in patients with papillary thyroid cancer.