Based on the mechanism of 2,4,6-Trinitrotoluene (TNT) degradation, an amperometric hydrogen peroxide biosensor was constructed for the determination of trace amounts of TNT by immobilization of MWCNTs, HRP and Nafion onto the surface of glassy carbon electrode (GCE). The Nafion/MWCNTs/HRP biosensor was capable of degrading TNT with the consumption of H2O2 and HRP in 0.2 mol/L PBS (pH 7.0). Trace TNT was quantitative analyzed by the current decrease of H2O2 at the reductive potential of 0.35V using cyclic voltammetry (CV). Effect of the ratio of MWCNTs/HRP, initial concentration of H2O2 and electrolyte's pH were also optimized by CV. Under the optimal conditions, the current decrease of H2O2 that was consumed by TNT degradation was proportional to TNT ranging from 8.8×10 9mol/L to 2.64×10-7 mol/L with a detection limit of 3.0×10-9 mol/L (S/N=3). It developed a new way for simple, rapid and sensitive measurement of trace TNT.