A highly sensitive method is presented for the colorimetric determination of malathion using gold nanoparticles (AuNPs). In this approach, the synthesized AuNPs solution was stabilized by the citrate anions as their repulsion protected the AuNPs from aggregation. The synthesized AuNPs were characterized morphologically by using transmission electron microscopy technique. Malathion caps the surface of AuNPs and induces the aggregation of AuNPs in Britton–Robinson buffer solution. The reaction was monitored spectrophotometrically by measuring the decrease in the plasmon resonance band of the AuNPs at 527 nm after 9 min. The effect of reaction variables on the reaction sensitivity was investigated and furthermore, the interference of common ions was effectively avoided. The calibration curve is linear over the concentration range 3.3 × 10−7 to 3.3 × 10−6 mol/L of malathion with good precision and accuracy and the detection limit was down to 1.5 × 10−7 mol/L. The developed approach does not use complex and expensive instruments. The high sensitivity of the proposed method allowed its successful application to wheat and water samples. Thus, the proposed strategy can serve as a powerful method for the rapid diagnosis of malathion in agriculture products.