In this work, nickel oxide nanoparticles‐modified multi‐walled carbon nanotubes (CNTs) were prepared and used for H2O2 sensing application. Firstly, ex situ NiO nanoparticles (NPs) were prepared and further used to decorate polyethylenimine (PEI)‐modified carboxylated CNTs. The obtained nanocomposite and its precursors were identified by using X‐ray diffraction, thermal analysis, Raman spectroscopy and SEM and TEM images, N2 adsorption‐desorption isotherms, and electrochemical techniques. The sensing properties of the NiO‐modified nanocomposite toward H2O2 were studied by electrochemical techniques using glassy carbon electrodes (GCEs) as support material. After optimizing the sensor construction, the sensor sensitivity was about of 0.83±0.01 A M−1 cm−2 with a LOD of about 1.0 μM. In addition, it showed excellent anti‐interference properties, reproducibility, and stability (over 4 months). Finally, such sensors were coupled to a flow injection device and the H2O2 concentration of some commercial antiseptic solutions were successfully obtained (with recovery ratios between 96.3–102.4 %).