A new electrochemical biosensor based on the immobilization of horseradish peroxidase(HRP) on TiO2 nanoneedles modified electrodes has been fabricated. The direct electrochemical response of HRP immobilized on the modified electrode was dramatically enhanced. The immobilized HRP displayed a couple of stable and well-defined redox peaks with a formal potential of -0.379 V (vs. SCE). The HRP/TiO2 nanoneedles modified electrode exhibited a remarkable electrocatalytic activity toward the oxidation of H2O2. The amperometric response to H2O2 showed a linear range of 4–700μmol/L, with the calculated detection limit of 0.78 μmol/L at a signal-to-noise ratio of three. The modified electrode displayed an acceptable reproducibility and good stability. The new HRP/TiO2 nanoneedles matrix is expected to have wide applications for enzymes and proteins immobilization and direct electron transfer study and opened a way for low conductivity electrode biosensors.