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 H2O2. The plot of the response current to H2O2 concentration exhibited a linear relation at a range of 3.0 x 10(- 6) to 4.3 x 10(- 4) M, with a correlation coefficient of 0.998. The detection limit is 4.3 x 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. (C) The Author(s) 2016. Published by ECS.