Down‐regulator of transcription 1 (DR1) is considered as a biomarker of hashimoto's thyroiditis (HT), which is a risk factor for thyroid cancer. Here, a label‐free electrochemical biosensor for DR1 detection was constructed based on polyamidoamine (PAMAM) polymer and the nanocomposite (WO3@AuNPs) composed of tungsten trioxide (WO3) and gold nanoparticles (AuNPs). WO3@AuNPs was obtained by combining monolayer WO3 nanosheets, which has high conductivity, and AuNPs. The modification of WO3@AuNPs can not only increase the conductivity of the electrode but also provide more active sites for signaling units, thus greatly improve the sensitivity of the sensor. The polymer PAMAM is biocompatible and non‐immunogenic, and its end functional group can bind to the target molecules, providing them with more binding sites and thus improving the sensitivity of the sensor. Under optimal conditions, the label‐free biosensor showed a good linear relationship between the logarithm of DR1 concentration and the impedance in the range of 10 fg·mL‐1 to 100 ng·mL‐1, with a detection limit as low as 0.3 fg·mL‐1. Besides, this label‐free electrochemical platform exhibited satisfactory selectivity and anti‐interference capability in human serum samples. Therefore, this method has considerable potential in clinical detection of DR1.