A renewable, disposable, low cost, and sensitive sensor for the detection of organophosphorus pesticides was constructed by immobilizing the acetylcholinesterase enzyme (AChE), via glutaraldehyde, on magnetic iron nanoparticles (Fe₃O₄) previously synthesized and functionalized with chitosan (CS). The sensor was denoted AChE/CS/Fe₃O₄. The magnetic nanoparticles were characterized by Fourier transform infrared spectroscopy and transmission electron microscopy. Acetylthiocholine (ATCh) was incubated with AChE/CS/Fe₃O₄ and attached to a screen-printed electrode using a magnet. The oxidation of thiocholine (from ATCh hydrolysis) was monitored at an applied potential of +0.5 V vs. Ag/AgCl(KCl_sat) in 0.1 mol L⁻¹ phosphate buffer solution (pH 7.5) as the supporting electrolyte. A mixture of the pesticide malathion and ATCh was investigated using the same procedure, and the results were compared and expressed as inhibition percentages. For determination of malathion, the proposed sensor presented a linear response in the range from 0.5 to 20 nmol L⁻¹ (R = 0.9942). The limits of detection (LOD) and quantification (LOQ) were 0.3 and 0.8 nmol L⁻¹, respectively. Real samples were also investigated, with recovery values of 96.0% and 108.3% obtained for tomato and pond water samples, respectively. The proposed sensor is a feasible option for malathion detection, offering a linear response, good sensitivity, and a low detection limit.