In this study, negatively charged poly (styrenesulfonate, sodium salt) (PSS) and positively charged poly (allylamine hydrochloride) (PAH) fibers have been alternatively assembled on the polystyrene (PS) surface by using layer-by-layer (LBL) technique. Specifically ordered films have then been prepared by the self-assembling of hemoglobin (Hb) on the surface of PS/(PAH/PSS)(4)/PAH or PS/ (PAH/PSS)(4) fibers. Our relevant studies illustrate that compared with Hb-PS/(PAH/PSS)(4)/PAH films, the self-assembled Hb-PS/ (PAH/PSS)(4) films could more efficiently facilitate the direct electron transfer of Hb on glassy carbon electrode (GCE), which may be attributed to the effect of the readily electrostatic interaction between Hb and PS/(PAH/PSS)(4). Besides, cyclic voltammetry of the assembled Hb-PS/(PAH/PSS)(4) films on GCE displays a quasi-reversible electrochemical behavior in pH 5.0 acetate buffers. The average formal potential of Hb heme Fe(III)/Fe(II) couples in Hb-PS/(PAH/PSS)(4) films is observed to shift linearly between pH 3.6 and 5.7 with a slope of -52.1 mV/pH, suggesting that one proton is coupled with each electron transfer. Moreover, our results indicate that the catalytic activity to the reduction of H2O2 could be observed on the Hb-PS/(PAH/PSS)(4) modified GCE. A linear relationship exists between peak current and H2O2 concentration from 2.5 x 10(-6) to 1.2 x 10(-4) M. Consequently, our observations demonstrate that this simple and convenient strategy of the assembling Hb-PS/(PAH/PSS)(4) On GCE could effectively facilitate the direct electrochemistry of relevant heme containing proteins, which can be further utilized as a promising sensitive biosensor to detect some specific biological substrate and related biological process. (C) 2008 Elsevier B.V. All rights reserved.