Different types of (polycation/polyanion), layer-by-layer (LBL) films were assembled on the surface of pyrolytic graphite (PC) electrodes by spin-coating, and then immersed in myoglobin (Mb) solutions at pH 5.0 to load Mb into the films. Compared with the traditional solution-dipping method in LBL assembly,the spin-coating approach was much more time saving but did not show substantial difference in Mb loading. The Mb-loaded LBL films demonstrated nearly reversible cyclic voltammetric (CV) responses for Mb Fe(III)/Fe(II) redox couple, and the surface concentration of electroactive Mb in the films (Gamma*, mol cm(-2)) could be used to compare the loading behaviors of Mb in different films. Among the six polyelectrolyte LBL films, {HECE/PSS}(n) films assembled with quaternized hydroxyethyl cellulose ethoxylate (HECE) and poly(styrenesulfonate) (PSS) demonstrated the largest Gamma* value for loaded Mb and the best performance in electrocatalysis toward hydrogen peroxide. CV, UV-vis spectroscopy and electrochemical impedance spectroscopy (EIS) were used to characterize the films and explore the reason why {HECE/PSS}(n)-Mb films showed better electrochemical response than the other Mb-loaded LBL films. The better understanding of the interactions involved in Mb loading may help us to find novel and more suitable protein-loaded LBL film systems in the future. (C) 2009 Elsevier Ltd. All rights reserved.