In this paper, we developed a new method for immobilization of horseradish peroxidase (HRP) on Nano-NiO/ MWCNTs/PANI composite film, which can effectively promote the direct electron transfer and electrocatalysis of enzyme in neutral solution. This composite film modified electrode was systematically investigated by altering the thickness of composite film, pH value of solution, and the mass ratio of MWCNT to NiO nanoparticles. The study showed that the effective immobilization of horseradish peroxidase on Nano-NiO/ MWCNTs/PANI was 30 cycles of cyclic voltammetry for the preparation of PANI film and the mass ratio of 10:1. The composite film possesses a high catalytic activity in pH 6.8 buffer solution containing H 2 O 2 . The plot of the response current to H 2 O 2 concentration exhibited a linear relation at a range of 3.0 × 10 −6 to 4.3 × 10 −4 M, with a correlation coefficient of 0.998. The detection limit is 4.3 × 10 −7 M(S/N = 3). These results indicated that the modified electrode was beneficial to retain the catalytic activity of the enzyme, and possessed good stability and reproducibility. The electrochemical technology has been most extensively applied in the determination of H 2 O 2 as an inexpensive and effective way. However, it is difficult to carry out slow reaction kinetics for many electrode materials in detecting hydrogen peroxide process, and is required a high overpotential to avoid interference of other compounds in the same potential region. Hence, the electrode was generally modified by polymer film to rapidly and sensitively measure H 2 O 2 . In the past research, polyaniline (PANI) has been widely investigated as a key modifying material in chemical and biological sensors 1-3 due to good environmental stability, facile synthesis, electrical and optical properties, and unique doping/de-doping behavior, 4,5 and its electrical property can be reversibly modulated by changing the state of oxidation. Additionally, it is suitable for PANI film due to a moderating effect for enzyme catalytic reaction signal and compatibility. 6,7 However, the conductivity of PANI depends on the pH values of solutions. Generally, the excellent redox characteristics are exhibited in acidic aqueous solutions at pH up to about 5, while a low conductivity can be found in a neutral solution and/or alkaline solutions. Therefore, the modified electrode with polyaniline cannot fabricate a high-sensitivity biosensor. Moreover, the low conductivity cannot effectively mediate electron transfer between redox enzymes and conductive supports in biosensor systems. To improve the conductivity of polyaniline film, a conductive material with high conductivity, such as carbon nanotube(CNT), is introduced into PANI matrix to form conducting polymer/carbon nanotube composites. 8,9 The addition of CNT promotes the electron transfer rate between film and electrode in this composite, and possesses high electroactivity in a neutral pH environment. [10][11][12] The combination between CNTs and polymer matrix could effectively improve sol...