Herein we report the preparation of a novel nanocomposite film containing zirconia nanoparticles (ZrO2 NPs), poly(toludine blue O) (PTBO) and gelatin functionalized multiwalled carbon nanotubes (GCNT). Gelatin has been used as a dispersing agent to prepare stable dispersion of MWCNTs in doubly distilled water. The prepared GCNT aqueous dispersion possesses long-term storage stability at room temperature. ZrO2 NPs and PTBO have been stepwise electrochemically deposited on the GCNT modified glassy carbon electrode (GCE) surface. The electrostatic interactions between the negatively charged ZrO2 NPs and positively PTBO provide good stability to the composite film. GCNT present in the composite film endorse fast electron transfer process. The maximum surface coverage concentration (Ґ) of PTBO was noticed at the nanocomposite film modified GCE, which is 0.324 x 10-9 mol cm-2. The reason for the high Ґ value of PTBO at the composite film could be ascribed to the larger surface area of GCNT and ZrO2 NPs. The composite film exhibits rapid electrocatalytic response in the linear H2O2 concentration range between 0.05 mM–0.25 mM, with a higher sensitivity of 82.13 μA μM-1 cm-2. Moreover, the proposed nanocomposite film detects H2O2 present in antiseptic solutions in good linear range, which shows its good practical applicability.