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Chlorpyrifos is a type of organophosphate insecticide used extensively in the plantation of tomatoes, a worldwide popular food. However, chlorpyrifos residues on tomatoes bring risks to human health considering the consumption level of tomato is relatively high across many countries. Accurate detection and strict regulation of chlorpyrifos residues are very important in controlling health risks related to chlorpyrifos. Therefore, we developed a rapid, highly sensitive, economical, and nondestructive surface-enhanced Raman spectroscopy (SERS)-based method that can quantitatively determine the content of chlorpyrifos in tomatoes. Silver colloid was optimized and used as the enhance substrate for SERS. Chlorpyrifos residues on tomato surface and in tomato extract were both measured with SERS, and the results acquired under these practical conditions well matched those of chlorpyrifos standard solutions. Linear relationships between chlorpyrifos concentrations and SERS intensities at crucial characteristic peaks under both conditions were established, and the results showed linearity across the entire measured range from 10−3 to 10−9 mol L−1. The detection limit of the proposed method was 10−9 mol L−1, which was much lower than the standards of chlorpyrifos use for China, Japan and EU, and was the lowest reported concentration chlorpyrifos had been measured on any fruit/vegetable surface or in its extract. The proposed method was very promising in the accurate, quantitative detection of pesticide residues, and it may be adapted to a variety of agricultural products and other areas in food science. [ABSTRACT FROM AUTHOR]