This paper reports on the synthesis of a novel nanocomposite of multi-wall carbon nanotubes (CNTs), magnetite nanoparticle (Fe3O4), and polyethylenimine (PEI) to construct an electrochemical sensor for highly sensitive determination of ciprofloxacin (CIP) drug in biological samples and pharmaceutical formulations. The nano composite contains many amine functional groups provided by PEI conductive polymer. Due to the high conductivity of CNTs and moderate conductivity of the polymer along with rich active sites of amine functional groups, the PEI@Fe3O4@CNTs nanocomposite displayed excellent electro-catalytic effect on the electro-oxidation of CIP. The nanocomposite was fully characterized using Fourier transform infrared spectroscopy, field emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, and transmission electron microscopy. For the electrochemical determination of CIP, the differential pulse voltammetry signal of the drug electro-oxidation on a glassy carbon electrode modified with the newly developed nanocomposite was investigated. Under the optimized experimental conditions, the electrochemical responses of the modified electrode were proportional to the drug concentrations in the range of 0.03-70.0 mu mol L-1 with a limit of detection 3.0 nmol L-1. Furthermore, the method was applied for the determination of CIP in the drug tablets, urine, and serum samples with acceptable recoveries (97-108) and satisfactory precisions (1-3%RSD).