A hemoglobin/silver nanoparticle heterolayer was fabricated for bioelectronic device with electrochemical signal-enhancement effect. As a device element, a hemoglobin, the metalloprotein, contained the heme group that showed the redox property was introduced for charge storage element. For electron transfer facilitation, a silver nanoparticle was introduced for electrochemical signal facilitation, the hemoglobin was immobilized onto Au substrate using chemical linker 6-mercaptohexanoic acid (6-MHA). Then, the silver nanoparticle was immobilized onto fabricated hemoglobin/6-MHA heterolayers by layer-by-layer (LbL) method. The surface morphology and surface roughness of fabricated heterolayer were investigated by atomic force microscopy (AFM). The redox property of hemoglobin/silver nanoparticle heterolayer was investigated by a cyclic voltammetry (CV) experiment for obtaining an oxidation potential and reduction potential. Moreover, for the assessing charge storage function, a chronoamperometry (CA) experiment was conducted to hemoglobin/silver nanoparticle-modified heterolayer electrode using oxidation and reduction potentials, respectively. Based on the results, the fabricated hemoglobin/silver nanoparticle heterolayer showed that an increased charge storage effect compared to hemoglobin monolayer-modified electrode.