A general methodology to prepare reagentless biosensor was developed based on self-assembly and electrodeposition. Redox active inorganic multilayers consisting of copper hexacyanoferrate (CuHCF) multilayers were formed by successive self-assembly. A simple and controllable electrodeposition approach was established for one-step fabrication of chitosan-enzyme layer on CuHCF modified electrode. Horseradish peroxidase was selected as the model enzyme. With CuHCF as the electroactive mediator, the developed reagentless biosensor exhibited a fast amperometric response for the determination of hydrogen peroxide (H₂O₂). The linear response ranged from 1.4 × 10^-5 to 2.0 × 10^-4 M with a detection limit of 1.2 × 10^-6 M. The biosensor exhibited high reproducibility and long-time storage stability. The proposed methodology could serve as a versatile platform for fabrication of electrochemical biosensors.