This report presents a highly sensitive, rhodamine B-covered gold nanoparticle (RB-AuNP) -based assay with dual readouts (colorimetric and fluorometric) for detecting organophosphorus and carbamate pesticides in complex solutions. The detection mechanism is based on the fact that these pesticides can inhibit the activity of acetylcholinesterase (AChE), thus preventing the generation of thiocholine (which turns the RB-AuNP solutions blue and unquenches the fluorescence of RB simultaneously). The color of the RB-AuNP solution remains red and the fluorescence of RB remains quenched. By use of this dual-readout assay, the lowest detectable concentrations for several kinds of pesticides including carbaryl, diazinon, malathion, and phorate were measured to be 0.1, 0.1, 0.3, and 1 μg/L, respectively, all of which are much lower than the maximum residue limits (MRL) as reported in the European Union pesticides database as well as those from the U.S. Department Agriculture (USDA). This assay allows detection of pesticides in real samples such as agricultural products and river water. The results in detecting pesticide residues collected from food samples via this method agree well with those from high-performance liquid chromatography (HPLC). This simple assay is therefore suitable for sensing pesticides in complex samples, especially in combination with other portable platforms.