This paper reports on the synthesis of a novel nanocomposite of multi-wall carbon nanotubes (CNTs), magnetite nanoparticle (Fe 3 O 4 ), 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 con- ductivity of CNTs and moderate conductivity of the polymer along with rich active sites of amine functional groups, the PEI@Fe 3 O 4 @CNTs nanocomposite displayed excellent electro-catalytic effect on the electro-oxida- tion 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 mi- croscopy. 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 in- vestigated. Under the optimized experimental conditions, the electrochemical responses of the modified elec- trode were proportional to the drug concentrations in the range of 0.03–70.0 μmol 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).