The recombinant carboxylesterase PvCarE1-catalyzed hydrolysis product p-nitrophenol (p-NP) can quench the fluorescence of glutathione-stabilized gold nanoclusters (GSH-AuNCs) through internal filtration effect, while organophosphate pesticides (OPs) can inhibit the PvCarE1 activity and maintain the fluorescence of GSH-AuNCs. Display omitted •We developed a sensitive fluorescence sensor for rapid organophosphate pesticide (OP) detection.•We utilized glutathione-stabilized gold nanoclusters (GSH-AuNCs) and recombinant carboxylesterase PvCarE1.•The fluorescence signal was quenched by p-nitrophenol (p-NP) without OPs.•OPs inhibit enzymes, thus maintaining GSH-AuNC fluorescence.•The method demonstrated high sensitivity for dichlorvos, trichlorfon, and profenofos. Considering the severe threat to human health posed by organophosphate pesticides (OPs), reliable and rapid detection methods are urgently needed. In this study, we developed a straightforward and sensitive fluorescence sensor for OP detection. The sensor is based on glutathione-stabilized gold nanoclusters (GSH-AuNCs) and the inhibition of the recombinant carboxylesterase PvCarE1. The fluorescence signal of GSH-AuNCs is efficiently quenched by p-nitrophenol (p-NP) through inner filter effect, which is the product of the recombinant carboxylesterase PvCarE1-catalyzed hydrolysis of p-nitrophenol acetate (p-NPA). In the presence of OPs, the activity of the recombinant carboxylesterase PvCarE1 is inhibited, resulting in decreased p-NP production, and the fluorescence signal of the GSH-AuNCs is maintained. The assay exhibited good sensitivity to dichlorvos, trichlorfon, and profenofos, with detection limits of 0.2 μg/L, 5 μg/L, and 5 μg/L, respectively. In addition, the developed assay was applied to apple sample detection, with recoveries ranging from 84.5 % to 106.3 % and RSDs ranging from 1.3 % to 10.5 %, demonstrating its high potential in monitoring OPs in food.