We provide a facile, cost-effective and environmentally friendly method to prepare 3D hierarchical porous Co3O4 film by employing eggshell membrane as bio-templates. The Co3O4 nanofibers interconnected with each other to construct a 3D hierarchical porous film with macropores and mesopores, which is more effective in facilitating reactants diffusion and the electron transfer. The developed sensor was applied for direct electrochemical non enzymatic detection of glucose. High sensitivity (366.03 mu A mM(-1) cm(-2)), fast response (3 s), anti-interference performance and excellent stability were achieved. The results demonstrate that the 3D hierarchical porous Co3O4 film have great potential applications in developing direct electrochemical non-enzymatic glucose biosensors. (C) 2016 Elsevier B.V. All rights reserved.