The nanocomposite of cobalt nanoflowers on graphene (Co-GE) was synthesized by a facile, one-pot ultrasonic electrochemical method. The scanning electron microscopy measurements displayed that the synthesized Co-GE exhibited a related hierarchical structure of a flake GE homogeneous distribution as a matrix for the growth of Co nanoflowers. The chemical composition was confirmed by energy dispersive X-ray spectrograms and X-ray diffraction analysis. The electrochemical biosensors based on redox proteins or enzymes possess high selectivity and biological compatibility but suffer from the low voltammetric response due to the deep burial of electroactive center in proteins. Herein, two typical denaturants, urea and acid, were synergistically utilized to maintain hemoglobin (Hb) in its most unfolded state, while simultaneously maintaining the presence of the heme groups. A novel hydrogen peroxide (H2O2) biosensor was structured using nanocomposite and protein conformational intermediate. The unfolded Hb/Co-GE/GCE exhibited accelerated direct electron transfer for sensing H2O2 under optimal conditions. The sensitivity for detecting H2O2 improved as much as 8.3 times higher than those for the native Hb/Co-GE/GCE. The electrocatalytic reduction of H2O2 showed a wide linear range from 0.25 to 190.0 mu M with a high sensitivity of 116.3 mu A mM(-1) and a low limit of detection of 0.08 mu M (S/N = 3). The unfolded Hb-based biosensor possesses the advantages of excellent stability, good reproducibility, and satisfactory recovery. The current research provides a novel strategy to utilize the unique properties of protein conformational intermediates in the development of electrochemical biosensors.