© 2021 Elsevier B.V.Herein, we synthesized ferromagnetic BiFeO3 nanoparticles with sizes of 20–60 nm and rhombohedral distorted perovskite structure without any co-formed impurity phases by sol-gel method. Structural, morphological, magnetic, electronic and textural properties of BiFeO3 nanoparticles were characterized by using XRD, SEM-EDX, TEM, FTIR, XPS, VSM, UV-DRS, PL and BET techniques. The peroxidase was isolated from the Cress (Lepidium sativum) and purified by using precipitation of ammonium sulfate and chromatographic techniques; gel filtration and ion exchange chromatography which is showed wide range of thermal stability, maximum activity in the acidic environment and close to the body temperature. Afterwards, a novel amperometric biosensor based on Cress peroxidase entrapped on BiFeO3 nanoparticles modified carbon paste electrode was prepared for the first time to sensitive detection of H2O2. The prepared H2O2 biosensor was operated with the aid of choro-amperometry technique by applying −0.8 V vs Ag/AgCl electrode. The developed biosensor exhibited a linear response towards to H2O2 in the concentration range of 2.0 10−7 to 1.0 10−5 M under the optimal conditions and limit of detection and quantification were estimated as 7.0 10−8 M (2.7 μg L−1) and 2.0 10−7 M (8.7 μg L−1), respectively. The repeatability of the electrode was calculated as 19.2 for 2.0 10−7 M (N = 7) and the accuracy was 81.2% for this concentration. In addition, the H2O2 biosensor exhibited excellent stability up to ten days. Finally, the sensor developed was used for the analysis of H2O2 in milk samples and satisfactory recoveries were obtained under the operating conditions.