Facile yet simple platforms for the immobilization of biomolecules have always been a substantial requirement for the fabrication of proficient biosensors. In this study, we report a naphthyl substituted acetate functionalized ionic liquid (NpAc-IL) for the covalent anchoring of horseradish peroxidase (HRP), using which the direct electrochemistry of HRP was successfully accomplished and a H2O2 biosensor was developed. The naphthyl substitution on the NpAc-IL was utilized for the π-π stacking with the MWCNT modified GCE and the terminal -OCH3 group of NpAc-IL was used for the covalent attachment with the free -NH2 group of HRP via amide bond formation. High conducting nature of the newly designed ionic liquid (NpAc-IL), facilitated an improved communication with the deeply buried redox centre of the HRP, while the covalent bonding provided enhanced stability to the fabricated biosensor by stably holding the water soluble HRP enzyme on the electrode surface. Furthermore, incorporation of MWCNT on the sensor setup synergistically enhanced the sensitivity of the developed biosensor. Under optimized conditions, the fabricated biosensor showed an enhanced electrocatalytic reduction of H2O2 in the range of 0.01 to 2.07 mM with a limit of detection and sensitivity of 2.7 μM and 55.98 μA mM−1 cm−2 respectively. Further, the proposed biosensor was utilized for the sensing of H2O2 spiked in real samples. Moreover, the newly fabricated biosensor demonstrated excellent stability with improved sensitivity and selectivity towards H2O2 reduction. The superior analytical characteristics are attributed to the facile fabrication strategy using this newly developed acetate functionalized ionic liquid platform. © 2020