Nonionic poly(ethylene glycol) (PEG) and myoglobin (Mb) were successfully assembled into {PEG/Mb}(n) layer-by-layer films on various solid surfaces. Quartz crystal microbalance (QCM), UV-vis spectroscopy and cyclic voltammetry (CV) were used to monitor and confirm the film growth and characterize the films. The Mb in stable {PEG/Mb}(n) films showed a quasi-reversible CV response for its heme Fe(III)/Fe(II) redox couple, and was used to electrocatalyze the reduction of various substrates. The interaction between PEG and Mb in the assembly was investigated in detail. A series of comparative experiments showed that the ion-dipole interaction between positively charged groups on the Mb surface and electronegative ether oxygen groups of PEG would be the main driving force for the assembly of {PEG/Mb}(n) films, while other interactions such as hydrogen bonding and/or hydrophobic interaction may also play an important role in stabilizing the films in blank buffers. (C) 2008 Elsevier Ltd. All rights reserved.