Highly porous hemoglobin (Hb) microbelts based biosensor was developed by directly electrospinning Hb onto the glassy carbon (GC) electrode surface without using immobilization matrix, offering an excellent electrochemical sensing platform. Ultraviolet-visible spectroscopy and Fourier transform infrared spectroscopy were performed to demonstrate that Hb still kept its native structure in the as-electrospun microbelts. The electrocatalytic property of Hb microbelts modified GC electrode was investigated using hydrogen peroxide (H(2)O(2)) and nitrite as model compounds. The cyclic voltammetry results have demonstrated that the Hb microbelts modified electrode shows enhanced activity in the electrochemical reduction of H(2)O(2) and nitrite, which offers a number of attractive features and is explored to develop an amperometric biosensor. The Hb microbelts based amperometric biosensor has fast responses to H(2)O(2) and nitrite, good dynamic response ranges, excellent detection limits of 0.61 mu M for H(2)O(2) and 0.47 mu M for nitrite (S/N = 3), and superior K(M,app) values of 0.093 mM for H(2)O(2) and 0.713 mM for nitrite, respectively. These results demonstrate that the electrospun Hb microbelts can significantly enhance the direct electrochemistry of Hb and has great potential application in mediator-free biosensor applications. (C) 2010 Elsevier B.V. All rights reserved.