An ultrasensitive sensor was developed for the detection of an organophosphorus compound using unmodified silver nanoparticles (AgNPs). The hydrolysis of acetylthiocholine (ATCh) by acetylcholinesterase (AChE) generates a thiol-containing compound, thiocholine, which influences the AgNP aggregation. Herein, we describe an indirect enzyme-based detection of malathion with AChE and ATCh in the presence of AgNPs using the fluorometric method. The addition of malathion to the system decreased the enzyme hydrolysis and thiocholine generation, and thus was able to quench the AgNPs, with a corresponding decrease in the emission intensity at 423nm (λex=340nm). The proposed system showed linearity in the range, 2-100fM, with an excellent limit of detection of 0.556fM by fluorescence spectroscopy. The aggregation and disaggregation of AgNPs were also supported by transmission electron microscopy and dynamic light scattering analyses. The potential application of the system was demonstrated by determination of malathion in real samples like agricultural runoff water, lake water, cabbage, and apple samples using fluorometric method, and the obtained results were further validated with HPLC. © 2015 Elsevier B.V.