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Hydrogen peroxide (H2O2) is a byproduct of oxidative metabolism, acts as a signaling molecule, and can induce protein and DNA damage by oxidative stress. Ionic liquid-coated lignin stabilized silver nanoparticles (LAgNPs) have been used for the colorimetric detection of H2O2. Both ionic liquid and lignin moieties have a synergistic effect on the conductivity of silver nanoparticles to display intrinsic peroxidase-like activity with the enhanced potential to catalyze the oxidation of substrate 3,3′,5,5′-tetramethylbenzidine (TMB). LAgNPs have been synthesized by the green synthesis method and the prepared nanoparticles have been characterized by different techniques such as FTIR, EDX, TGA, SEM, and XRD. 1-H-3-methylimidazolium acetate (ionic liquid) was prepared and used for tuning of AgNPs. By using ionic liquid capped LAgNPs the colorimetric detection of H2O2 was done with the assistance of TMB solution and results were analyzed by UV–Vis spectrophotometer. Different parameters such as (amount of capping agent and TMB, pH, H2O2 concentration, and time) were optimized to get the best results of the proposed sensor. The H2O2 biosensor exhibited a linear response in the range of 1 × 10−9–3.6 × 10−7 M, with a detection limit of 1.379 × 10−8 M and a quantification limit of 4.59 × 10−8 M, and R2 of 0.999. The sensor gave a short response time of 5 min for colorimetric detection of H2O2 at pH 7.5 and room temperature. For the detection of hydrogen peroxide, the proposed sensor showed good sensitivity and selectivity and was successfully employed for the detection of H2O2 in the blood serum samples of hypertension patients.