Estrogen receptor alpha (ERα) is an important biomarker for breast cancer, appearing in over 75% of breast cancer cases. In this study, an electrochemical biosensor was constructed for sensitive detection of ERα based on the signal amplification of AuNPs-SWCNT nanocomposites and hemin@HKUST-1. AuNPs-SWCNT was used as a conductive substrate to immobilize the aptamer (Apt) by an Au-S bonding. Then, Apt, ERα, and antibody (Ab) formed a sandwich composite structure through specific recognition and immobilized on the electrode. The hemin@HKUST-1 was introduced into the biosensor via an amide bond with Ab and acted as the signal molecule. AuNPs-SWCNT could significantly enhance the conductivity of the electrode and the large specific surface area of HKUST-1 enabled it to load more hemin, and greatly improved the sensitivity of the biosensor. The detection limit of the biosensor could reach 27.8 fg·mL-1 after optimization of the conditions, with the current intensity and the logarithm of the ERα concentration exhibited a good linear relationship between 100 fg·mL-1 and 100 ng·mL-1. In addition, the biosensor possessed excellent selectivity, stability, reproducibility, and interference immunity. More importantly, it shows great application potential in practical detection.