Most of the fluorescence sensors require choline oxidase or quenchers to detect organophosphorus pesticides (OPs) based on a single hydrolysate and suffer from high cost, complex procedures, weak stability, and low sensitivity. Here, we proposed a brand-new fluorescence strategy for highly sensitive detection of OPs based on both hydrolysate-response disulfide bond-functionalized gold nanoclusters (S-S-AuNCs) without additional substances. S-S-AuNCs were synthesized via a facile one-step redox reaction and emitted bright red light with ultrasmall size and high water dispersion. Interestingly, S-S-AuNCs displayed a unique response to thiol compounds and low pH values and were thus pioneered as a high-efficiency sensor for OPs based on acetylcholinesterase (AChE)-catalyzed hydrolysis of acetylthiocholine into thiocholine and CHCOOH and OP inhibition of AChE activity. Further, S-S-AuNCs were employed to monitor the residue, distribution, and metabolization of methidathion in pakchoi with acceptable results. We believe that this work supplies a simpler and more highly sensitive approach for OP assay than the known ones and opens a new avenue to development of multistimulus-responsive and high-performance fluorescence substances.