A cationic quinine-imide dye brilliant cresyl blue (BCB) and horseradish peroxidase (HRP) were co-immobilized within ormosil on multiwalled carbon nanotubes modified glassy carbon electrode for the fabrication of highly sensitive and selective hydrogen peroxide biosensor. The presence of epoxy group in ormosil as organic moiety improves the mechanical strength and transparency of the film and amino group provides biocompatible microenvironment for the immobilization of enzyme. The presence of MWCNTs improved the conductivity of the nanocomposite film. The surface characterization of MWCNT modified ormosil nanocomposite film was performed with scanning electron microscopy (SEM) and atomic force microscopy (AFM). Cyclic voltammetry and amperometry measurements were used to study and optimize the performance of the resulting peroxide biosensor. The apparent Michaelis–Menten constant was determined to be 1.5 mM. The proposed H₂O₂ biosensor exhibited wide linear range from 3×10⁻⁷ to 1×10⁻⁴ M, and low detection limit 1×10⁻⁷ M (S/N=3) with fast response time <5 s. The probable interferences in bio-matrix were selected to test the selectivity and no significant response was observed in the biosensor. This biosensor possessed good analytical performance and long term storage stability.