A whole-cell microbial amperometric biosensor based on toluene-o-xylene monoxygenase (ToMO), expressed by Escherichia coli TG1 harboring pBS(Kan)ToMO was constructed. After that, the biosensor was characterized for the detection of aromatic hydrocarbons such as phenol, p-cresol, catechol, and pyrogallol. Chitosan and Fe3O4 magnetic nanoparticles were used as the bionanocomposite matrix to immobilize the bacterial cells in the biosensor construct. The electron transfer rate enhanced by the bio-catalytic activity of the magnetic nanoparticles and possession of the porous morphology of chitosan allowed efficient entrapment of the bacterial cells. Amperometric detection was based on the hydroxylation reaction catalyzed by ToMO, which resulted on substrate oxidation process. The electrochemical behaviors of the biosensor were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The prepared enzyme electrodes exhibited maximum response on catechol at pH 7.4 and 40 °C, low applied potential of +0.35 V, with sensitivity of 3.0 μA · μm –1, linear range of 1–10 μm and low detection limit of 0.5 μm. The ToMO based biosensor exhibited a better response to aromatic hydrocarbons with the addition of Fe3O4 and with the use of polymeric mediator film, which played an important role in the biosensor performance.