A novel hydrogen peroxide biosensor has been designed based on the co-immobilization of hemoglobin (Hb). TiO2 nanotubes and methylene blue (MB) onto a glassy carbon (GC) electrode. The titania nanotube arrays were directly grown on a Ti substrate using anodic oxidation first; and then detached from the Ti substrate using ultrasonication. A pair of well-defined redox peaks of Hb, enhanced by MB, appeared at an applied potential around 0.425 V. revealing that the immobilized Hb exhibits direct electrochemistry. Our electrochemical study shows that the immobilized Hb remains highly bioactive and displays excellent electrocatalytic response toward H2O2. The effect of pH and applied electrode potential in H2O2 sensing has been systemically studied. Besides the advantages of fast response and high stability, under the optimized conditions the linearity of the developed biosensor for the detection of H2O2 was observed from 2 x 10(-7) to 1.6 x 10(-4) M with a detection limit as low as 8 x 10(-8) M (based on S/N = 3). (C) 2011 Elsevier B.V. All rights reserved.