A highly sensitive electrochemical biosensor with a signal amplification platform of electrodeposited gold nanoparticle (AuNP) has been developed and characterized. The sizes of the synthesized AuNP were found to be critical for the performance of biosensor in which the sizes were dependent on HAuCl₄ and acid concentrations; as well as on scan cycles and scan rates in the gold electro-reduction step. Systematic investigations of the adsorption of proteins with different sizes from aqueous electrolyte solution onto the electrodeposited AuNP surface were performed with a potentiometric method and calibrated by design of experiment (DOE). The resulting amperometric glucose biosensors was demonstrated to have a low detection limit (> 50M) and a wide linear range after optimization with AuNP electrodeposition.