Three-dimensional (3D) graphene with high specific surface area, excellent conductivity and designed porosity is essential for many practical applications. Herein, holey graphene oxide with nano pores was facilely prepared via a convenient mild defect-etching reaction and then fabricated to 3D nanostructures via a reduction method. Based on the 3D architectures, a novel enzymatic hydrogen peroxide sensor was successfully fabricated. Transmission electron microscopy (TEM), scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) were used to characterize the 3D holey graphene oxide architectures (3DHGO). Cyclic voltammetry (CV) was used to evaluate the electrochemical performance of 3DHGO at glassy carbon electrode (GCE). Excellent electrocatalytic activity to the reduction of H₂O₂ was observed, and a linear range of 5.0×10-8~5.0×10-5 M with a detection limit of 3.8×10-9 M was obtained. These results indicated that 3DHGO have potential as electrochemical biosensors.