Oppositely charged poly(allylamine hydrochloride) (PAH) and 5nm-sized TiO2 nanoparticles were assembled into {PAH/TiO2} n layer-by-layer (LBL) films on electrodes. The cyclic voltammetric (CV) response of K3Fe(CN)6 at {PAH/TiO2} n film electrodes was very sensitive to the solution pH. At pH 4.0, the probe showed a nearly reversible CV peak pair with large peak currents; at pH 9.0, however, the CV response was significantly suppressed. This reversible pH-dependent on–off property could be used to switch the bioelectrocatalysis of H2O2 by horseradish peroxidase (HRP) with Fe ( CN ) 6 3 - as the mediator by changing the surrounding pH. The mechanism of the pH-dependent permeability of the films toward the probe was explored and discussed by comparative experiments.