A uniform bionanomultilayer constructed with soluble multiwall carbon nanotubes (MWNTs) was developed. The excellent solubility of MWNTs in aqueous solutions was achieved for the first time simply through the function of barbiturates without having recourse to surfactant or polymer. The soluble MWNTs were selected to construct the bionanomultilayer of MWNTs and horseradish peroxidase (MWNTs/HRP), via layer-by-layer assembly. UV-vis spectra, scanning electron microscopy, Raman spectra and atomic force microscopy reveal the uniform assembly process and the homogeneity of the developed bionanomultilayer. The bionanomultilayer biosensor can be successfully applied to detect hydrogen peroxide, which presented a linear response for hydrogen peroxide from 0.4 to 12.0 mu M with a detection limit of 0.08 mu M. The MWNTs in the biosensor provided a suitable microenvironment to retain HRP activity and acted as a transducer for improving the electron transfer and amplifying the electrochemical signal of the product of the enzymatic reaction. So the developed bionanomultilayer biosensor exhibited a fast, sensitive and stable response. (c) 2008 Elsevier B.V. All rights reserved.