In this study, a highly sensitive fluorescent probe based on bovine serum protein-protected gold nanoclusters (BSA-AuNCs) was developed for the determination of organophosphorus pesticides (OPs). At first, BSA-AuNCs were prepared using a photo stable fluorescent substrate. Acetylcholinesterase (AChE) catalyzes the hydrolysis of acetylthiocholine iodide (ATCI) to produce thiocholine (TCh), and the sulfhydryl groups (SH–) on TCh form Au–S bonds with BSA-AuNCs; thus, the fluorescence of BSA-AuNCs weakens. On the other hand, OPs can inhibit the activity of AChE, thus preventing the generation of TCh, and the fluorescence recovery of BSA-AuNCs occurs. Under optimized experimental conditions, parathion-methyl (PM) was detected in the concentration range of 0.33–6.67 ng mL⁻¹ with a detection limit of 0.14 ng mL⁻¹ (S/N = 3), which is much lower than the maximum residue limits reported in the European Union pesticides database as well as that defined by the U.S. Department Agriculture. Moreover, the assay demonstrated highly sensitive applications in the quantitative determination of OPs in water and food samples.