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A poly(N-isopropylacrylamide) (PNIPAm) brush has been successfully fabricated on an indium tin oxide (ITO) film via a simple electrochemical route. The polymer thermoresponsive behavior was investigated with an electrically heated ITO electrode. This kind of electrode demonstrated a rapid response to heating up and down, and the results indicated that the polymer modified interface possessed a characteristic lower critical solution temperature (LCST) and showed ON/OFF switch behavior. Furthermore, the model protein of hemoglobin (Hb) was incorporated into the polymer by a thermal "breathing-in" process. The electrochemical experiments revealed that the film could provide a friendly microenvironment for Hb to promote direct electron transfer. A pair of well-defined redox peaks with a formal potential of -204 mV (verses saturated calomel electrode, SCE) was observed. The redox current of Hb gradually lowered when the prepared electrode was immersed into pH 7.0 PBS at 20 °C, indicating that incorporated Hb could be released from the PNIPAm film, which proved that the approach could provide a potential route in the design of responsive biocompatible surfaces.