Nanostructured composite films of multiwalled carbon nanotube/polypyrrole (MWCNT/PPy) were prepared, and their potential as potentiometric biosensors was studied. MWCNT and PPy were alternately assembled on indium tin oxide (ITO) electrodes via a solution-based layer-by-layer self-assembly technique, which provided a good matrix for the immobilization of horseradish peroxidase (HRP). The resulting MWCNT/PPy/HRP composite films exhibited excellent potentiometric response to hydrogen peroxide while the thickness and composition of the multilayer were found to affect the performance of the biosensor.