Dual-responsive poly(N,N-diethylacrylamide) (PDEA) hydrogel films with entrapped horseradish peroxidase (HRP) and glucose oxidase (GOD) were successfully prepared on electrode surface with a simple one-step polymerization procedure under mild conditions, designated as PDEA-HRP-GOD. Cyclic voltammetric (CV) response of electroactive probe K3Fe(CN)(6) at the film electrodes displayed reversible thermo- and sulfate-responsive switching behavior. For example, at 25 A degrees C, the K3Fe(CN)(6) demonstrated a well-defined CV peak pair with large peak currents for the films, showing the on state, while at 40 A degrees C, the CV response was greatly suppressed and the system was at the off state. The influence of temperature and Na2SO4 concentration on the switching behavior of the film system was not independent or separated, but was synergetic. The responsive mechanism of the system was ascribed to the structure change of PDEA component in the films with temperature and sulfate concentration. This switching property of the PDEA-HRP-GOD films could be further used to realize dual-responsive catalytic oxidation of glucose sequentially by HRP and GOD entrapped in the films with Fe(CN)(6) (3-) as the mediator through changing the surrounding temperature and Na2SO4 concentration. This system may establish a foundation for fabricating a new type of multi-switchable electrochemical biosensors based on bienzyme electrocatalysis.