Creation of interfaces with a prudent design for the immobilization of biomolecules is substantial in the construction of biosensors for real-time monitoring. Herein, an adept biosensing interface was developed using a nanoconjugated matrix and has been employed toward the electrochemical determination of hydrogen peroxide (H2O2). The anionic gold nanoparticle (AuNP) was electrostatically tethered to cationic redox ionic liquid (IL), to which the horseradish peroxidase (HRP) enzyme was covalently immobilized to form a nanobioconjugate. The anthracene-substituted, aldehyde-functionalized redox IL (CHO-AIL) was judiciously designed with the (i) imidazolium cation for electrostatic interaction with AuNPs, (ii) anthracene moiety to mediate the electron transfer, and (iii) free aldehydic group for covalent bonding with a free amine group of the enzyme. Thus, the water-soluble HRP is effectively bonded to the CHO-AIL on a glassy carbon electrode (GCE) via imine bond formation, which resulted in the formation of the HRP-CHO-AIL/GCE. Electrochemical investigations on the HRP-CHO-AIL/GCE reveal highly stable and distinct redox peaks for the anthracene/anthracenium couple at a formal potential (E°′) of-0.47 V. Electrostatic tethering of anionic AuNPs to the HRP-CHO-AIL promotes the electron transfer process in the HRP-CHO-AIL/AuNPs/GCE, as observed by the reduction in the formal potential to-0.42 V along with the enhancement in peak currents. The HRP-CHO-AIL/AuNPs/GCE has been explored toward the electrocatalytic detection of H2O2, and the modified electrode demonstrated a linear response toward H2O2 in the concentration range of 0.02-2.77 mM with a detection limit of 3.7 μM. The developed biosensor ascertained predominant selectivity and sensitivity in addition to remarkable stability and reproducibility, corroborating the suitableness of the platform for the effectual biosensing of H2O2. The eminent performance realized with our biosensor setup is ascribed to the multifunctional efficacy of this newly designed nanobioconjugate. © 2020 American Chemical Society.