The wide use of pesticides in agriculture makes necessary the development of cost-effective, easy, and rapid methods aiming for environmental and health protection. We propose the fabrication and investigation of layer-by-layer (LbL) films composed of reduced graphene oxide stabilized in polyelectrolytes in the presence and absence of gold nanoparticles (AuNPs) toward methyl parathion (MP) detection. In addition to the film assembly and sensing application, we performed a systematic investigation of the influence of MP distribution on the limit of detection and linear range of the sensor. Indium tin oxide (ITO) electrodes modified with the LbL films were applied by differential pulse voltammetry achieving an LOD and a linear range of 0.226 ppm and 0.25–40 ppm in the absence of AuNPs, while in the their presence, a wider linear range was achieved (0.5–60 ppm, with LOD of 0.770 ppm). Real sample analysis indicated potential application in water, ground, and vegetables, with recovery varying from 92% to 113%.