Prussian blue nanoparticles (PBNPs) have peroxidase‐like activity for H2O2. However, PB alone have poor electrochemical performances. Herein, a strategy was proposed by direct in‐situ growth PBNPs onto gold nanowires (AuNWs) surface to obtain the peroxidase‐like activity with about 4.05 times higher than that of PBNPs alone. PBNPs@AuNWs was employed to construct a non ‐ enzymatic electrochemical H2O2 sensor with the detection limit of 5.3×10−9 mol/L (S/N=3). The sensor was successfully used to detect H2O2 in human serum samples or secreted from living HeLa cells. It may be a competitive candidate for H2O2 assaying in biological samples or cellular investigation.