Fabrication of high‐performance surface‐enhanced Raman scattering (SERS) biosensors relies on the coordination of SERS substrates and sensing strategies. Herein, a SERS active AuCu alloy with a starfish‐like structure is prepared using a surfactant‐free method. By covering the anisotropic AuCu alloy with graphene oxide (GO), enhanced SERS activity is obtained owing to graphene‐enhanced Raman scattering and assembly of Raman reporters. Besides, stability of SERS is promoted based on the protection of GO to the AuCu alloy. Meanwhile, it is found that SERS activity of AuCu/GO can be regulated by DNA. The regulation is sequence and length dual‐dependent, and short polyT reveals the strongest ability of enhancing the SERS activity. Relying on this phenomenon, a SERS biosensor is designed to quantify apurinic/apyrimidinic endonuclease 1 (APE1). Because of the APE1‐induced cycling amplification, the biosensor is able to detect APE1 sensitively and selectively. In addition, APE1 in human serum is analyzed by the SERS biosensor and enzyme‐linked immunosorbent assay (ELISA). The data from the SERS method are superior to that from ELISA, indicating great potential of this biosensor in clinical applications.
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