The construction of anti-fouling biosensing interface with inherent electroactivity is helpful to improve the practicability of the electrochemical biosensors. Herein, an electroactive biosensing interface with excellent biofouling resistance ability has been developed based on a hybrid membrane of Ce3+-poly(dopamine) (PDA) and Prussian blue (PB). The PB that electrosynthesized on the glassy carbon electrode (GCE) endows the electrode intense electrochemical signals. The film of self-polymerized PDA with the coordinated Ce3+ that covers on the PB not only enhances the hydrophilicity as well as biofouling resistance ability of the interface, but also acts as the matrix for immobilization of -PO43− tagged probe DNA. Upon the hybridization with the target miRNA Let-7a to form a duplex structure, the micro-environment at the electrode–electrolyte interface is altered, resulting in the signal attenuation of PB. The miRNA Let-7a can be detected in a wide concentration range from 0.1 fM to 10 nM with a detection limit of 0.03 fM. Owing to the excellent anti-fouling ability of hybrid film, the biosensor is applicable for miRNA Let-7a analysis in the human serum samples. This study provides a new idea to construct the multi-functional biosensing interface to enhance their practical application potential.