MicroRNA, as a distinctive biomarker, plays a crucial role in the early prognosis and diagnosis of numerous severe diseases. However, due to its inherent properties such as low abundance, small size, and high sequence similarity, the sensitive and accurate detection of microRNA remains a major challenge. Herein, a dual-mode electrochemical biosensing platform was developed for microRNA detection, based on poly(3,4-ethylenedioxythiophene) (PEDOT) doped with graphene oxide-Fe3O4 (GO-Fe3O4) nanocomposite. The GO-Fe3O4/PEDOT composite demonstrated a porous microstructure, outstanding conductivity, and robust catalytic activity towards nitrite. It was electrodeposited onto the electrode surface in a one-step process using the cyclic voltammetry method (CV). The microRNA biosensor was obtained by anchoring DNA with amino groups to the GO-Fe3O4/PEDOT layer through the formation of amide bonds. The designed dual-mode microRNA biosensor demonstrated a broad linear range spanning from 10−15 M to 10−6 M, with low detection limits of 5.18 × 10−15 M and 7.36 × 10−15 M when using chronocoulometry (CC) and amperometric i-t curve (i-t) modes, respectively. Furthermore, a dual-mode electrochemical biosensor has been successfully developed and utilized for the detection of microRNA in human serum, demonstrating its potential for precise and sensitive microRNA detection and its practical application value in clinical medicine.