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In this study, we have developed a sensitive approach to measure organophosphorus pesticides (OPs) using graphitic-phase C3N4 nanosheets (g-C3N4) combined with a nanomaterial-based quencher, MnO2 nanosheets (MnO2 NS). Since MnO2 NS can quench the fluorescence of g-C3N4 via the inner-filter effect (IFE), enzymatic hydrolysate (thiocholine, TCh) can efficiently trigger the decomposition of MnO2 nanosheets in the presence of acetylcholinesterase (AChE) and acetylthiocholine (ATCh), resulting in the fluorescence recovery of g-C3N4. OPs, as inhibitors to AChE activity, can prevent the generation of TCh and decomposition of MnO2 nanosheets while exhibiting fluorescence quenching. Therefore, the AChE-ATCh-MnO2-g-C3N4 system can be utilized to quantitatively detect OPs based on g-C3N4 fluorescence. Under optimal conditions, the linear ranges for the determination of parathion-methyl (PM) and 2,2-dichlorovinyl dimethyl phosphate (DDVP) were found to be 0.1–2.1 ng/mL and 0.5–16 ng/mL, respectively, with limits of detection of 0.069 ng/mL and 0.20 ng/mL, respectively. The advantages of this assay are user-friendliness, ease of use, and cost effectiveness compared to other more sophisticated analytical instruments. [ABSTRACT FROM AUTHOR]