Magnetic nanoparticles have been applied in many areas of nanomedicine. Sensing platforms based on this type of nanoparticles have received attention due to the relative low cost and biocompatibility. Biosensor is the most widely investigated type of analytical device. This type of sensor combines the physicochemical transduction with the incorporation of biological sensing components. Among the biological components, enzymes are the most commonly used as sensitive elements. However, natural enzymes may exhibit serious disadvantages such as lack of stability and loss of catalytic activity after immobilization. The study of enzymatic biomimetic systems are of great interest. This study reports the development of a new sensor composed of Fe3O4@CTAB and poly(sodium 4-styrenesulfonate) (PSS) films assembled via Layer-by-Layer (LbL) technique and used as peroxidase mimetic systems. Magnetic nanoparticles (MNps) were synthesized using thermal decomposition method and further dispersed to aqueous medium by ligand modification reaction using cetyltrimethylammonium bromide (CTAB). The amperometric detection limit of H2O2 was found to be ca. 103 μmol L-1. By chronoamperometry, the peroxidase biomimetic sensor exhibited a linear response for H2O2 in the range from 100 μmol L-1 to 1.8 mmol L-1 (R2 = 0.994) with sensitivity of 16 nA mol-1 L. The apparent Michaelis-Menten constant was 5.3 mmol L-1, comparable with some biosensors based on peroxidase enzyme. Moreover, the sensor presented a reproducibility of ca. 7.7% (n = 4) and their response (response time: 90 s) is not significantly affected in the presence of some interferents including K+, Na+, Cl-, Mg2+, Ca2+, and Uric Acid.