Electrochemical sensing of 4-acetaminophen by nickel oxide modified glassy carbon electrode was explored. Nickel oxide was prepared through simple hydrothermal method. The physical characterizations of nickel oxide nanoparticle were carried out by using advanced characterization techniques to investigate the phase transformation, surface morphology and functional groups present in the nickel oxide nanoparticle. Characterization techniques such as X-ray diffraction spectroscopy, ultra violet-visible spectroscopy, Fourier transform infra-red spectroscopy, Raman spectroscopy, X-ray photoelectron spectroscopy, field emission scanning electron microscopy and high resolution transmission electron spectroscopy were used. Particularly, high resolution transmission electron microscopy studies revealed particle size in the range of 15–20 nm. Herein, we report nickel oxide modified electrode for the detection of 4-acetaminophen under neutral condition using phosphate buffer solution (pH = 7). The nickel oxide modified electrode was used to detect 4-acetaminophen by cyclic voltammetry, differential pulse voltammetry and chronoamperometry techniques. The oxidation peak potential of 4-acetaminophen was observed at 0.35 V. The sensitivity and limit of detection for the determination of 4-acetaminophen were 91.0 μA cm−2 mM−1 and 0.23 μM, respectively. The sensor also demonstrates wide detection range (7.5–3000 μM), good stability, selectivity and antifouling properties. The practical applicability of the proposed sensor was successfully demonstrated for the determination of 4-acetaminophen in different human serum real samples and paracetamol tablets.