The emerging pollutant N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6PPD-quinone) has attracted broad attention because of its widespread presence and harmful impacts, including hepatotoxicity and neurotoxicity. Acetylcholinesterase (AChE) is commonly used as a classical biomarker for assessing toxicity in the nervous system. Here, the interaction mechanism between AChE and 6PPD-quinone was investigated using a combination of multispectral and computational approaches, including enzyme activity assay, fluorescence thermodynamic titration, circular dichroism (CD) spectroscopy, molecular dynamics (MD) simulation, computational alanine scanning (CAS), and free energy landscape (FEL) analysis, among others. The result indicates that 6PPD-quinone spontaneously binds into the active site of AChE, thereby competitively inhibiting enzyme's activity. The interaction is primarily facilitated by hydrogen bonds and van der Waals forces, exhibiting a binding constant (K