Triple-responsive films with binary architecture for switchable biosensors were designed on electrodes through the combination of the weak polyelectrolyte {PAH/PAA}(n) (PAH = poly(allylamine hydrochloride), PAA = poly(acrylic acid)) layer-by-layer assembly and chemically polymerized poly(N,N-diethylacrylamide) (PDEA) hydrogel containing horseradish peroxidase (HRP). The films exhibited reversible pH-, thermo-, and sulfate concentration responsively reversible on-off behaviors toward K3Fe(CN)(6) in its cyclic voltammetric (CV) response. Taking thermo-switch as an example, at 25 degrees C, CVs showed a large and well-defined peak pair of Fe(CN)(6)(3-) and the system was at the on state; while at 40 degrees C, the CV peaks were greatly suppressed and the system was at the off state. The bioactivity of HRP enzyme embedded in the films was well maintained and this film system could be further employed to control and modulate the bioelectrochemical reduction of H2O2 by HRP with K3Fe(CN)(6) as the mediator in solution. This triple-triggered bioelectrocatalysis based on the binary interface system could be used as a 3-input AND logic gate and demonstrated the potential perspective for fabricating novel multiple factor-switchable biosensors with immobilized enzymes. (C) 2016 The Electrochemical Society. All rights reserved.