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Herein, a sensitive sensing platform for acetaminophen based on palladium and multi-walled carbon nanotube nanocomposites and its electrochemical detection mechanism were presented in the first time. Transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy were utilized to characterize the morphology and structure of Pd-MWCNTs composite. Through the computer simulation, the Gaussian 09 and Multiwfn methods were used to study the redox mechanism of AP. Further electrochemical performance of AP was carried out by cyclic voltammetry (CV). The experimental results illustrated that Pd-MWCNTs modified electrode amplified the redox current of AP. Quantitative detection of AP by the DPV method revealed that the oxidation peak current of AP showed an acceptable linear response of 0.5 μM–100 μM with a detection limit of 0.13 μM (S/N = 3). The modified electrode also exhibited a good recovery in real sample detection. Image 1028 • An original and sensitive method for the determination of acetaminophen was developed. • Palladium/multi-walled carbon nanotubes composite (Pd/MWCNTs) was utilized to modify electrode. • A new type of sensor was obtained by a facile process with dropping Pd/MWCNTs on a glassy carbon electrode. • A novel reaction mechanism of acetaminophen was proposed by introducing computer calculation. [ABSTRACT FROM AUTHOR]