In this work, a nanocomposite based on nickel ferrite/activated carbon (NiF/AC) was used to modify a highly sensitive electrochemical sensor for the quantification of theophylline (TPL) in pharmaceutical tablets. The synthesized materials were characterized using x-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, energy dispersive x-ray spectroscopy-elemental mapping and surface area analysis via the Brunauer–Emmett–Teller method. Cyclic voltammetry was employed to study the electrocatalytic properties of the NiF/AC-GCE toward the oxidation of TPL. The dependence of the electrochemical response on the scan rate and pH was also investigated, and the working parameters were optimized. The linear range of the established electrochemical biosensor was from 0.5 to 5 μM (R2 = 0.997), with a detection limit of 0.21 μM. The present method was tested using three pharmaceutical formulation standard samples with good accuracy and acceptable recovery. Thus, it is a promising candidate for the determination of TPL in pharmaceutical formulations.