Horseradish peroxidase was immobilized on a glass carbon electrode by poly ( N-isopropylacyamide- co-3-methacryloxy-propyl-trimethoxysilane) (PNM). Horseradish peroxidase entrapped in the PNM film exhibited a pair of well-defined, quasi-reversible cyclic voltammetric peaks at about −0.36 V versus a saturated calomel electrode (SCE) in pH 7.0 buffer solution, corresponding to hemeFe III + e → hemeFe II. Some electrochemical parameters were calculated by performing nonlinear regression analysis of square wave voltammetry (SWV) experimental data. Fourier transform infrared (FTIR) spectra suggested that horseradish peroxidase entrapped in the PNM film retained the secondary structure. Hydrogen peroxide could be reduced by the catalysis of the entrapped horseradish peroxidase without any mediator. The reagentless hydrogen peroxide sensor had a fast response of less than 2.5 s with linear range of 0.19–1.35 μM with a detection of 4.75 × 10 −8 mol L −1. The sensitivity of the sensor for H 2O 2 was 0.62 A mol −1 cm −2. The activation energy for enzyme reaction was calculated to be 14.84 kJ mol −1.