Owing to the importance of hydrogen peroxide (H2O2) in environment and biological systems, selective and sensitive detection of this key analyte has gained a large interest in the area of electroanalytical chemistry. Herein, we have synthesized highly water-insoluble phosphonium based carboxyl functionalized ionic liquid (TPP-HA[TFSI]) and characterized using various spectral techniques and single crystal X-ray diffraction. The synthesized TPP-HA[TFSI] was deposited over a multiwalled carbon nanotube (MWCNT) coated glassy carbon electrode (GCE). Thus obtained TPP-HA[TFSI]/MWCNT/GCE was used as a host matrix for the covalent immobilization of water soluble protein cytochrome c (Cyt c) through EDC/NHS coupling reaction to form a stable amide bond between free [sbnd]COOH groups of TPP-HA[TFSI] and -NH2 groups of Cyt c (Cyt c/TPP-HA[TFSI]/MWCNT/GCE). Electrochemical characterization of the fabricated electrode by cyclic voltammetry revealed distinct redox peaks corresponding to the heme active sites of Cyt c (FeIII/FeII) with a cathodic and anodic peak at −0.39 V and −0.27 V. Further, the modified electrode exhibited excellent performance towards electrochemical reduction of H2O2. A stable catalytic response was observed at an operating potential of −0.45 V using amperometry. The linear range, sensitivity and detection limit for the determination of H2O2 at Cyt c/TPP-HA[TFSI]/MWCNT/GCE were found to be 20 to 892 μM, 0.14 μA μM−1 cm−2 and 6.2 μM, respectively. Notably, the proposed modified electrode demonstrated excellent selectivity and sensitivity towards the determination of H2O2 along with remarkable stability and reproducibility. © 2019 Elsevier B.V.