Recent studies have conclusively show that the surface-enhanced Raman spectroscopy (SERS) has been widely used for chemical and biomolecular sensing. In this work, a facile, low-cost, green and rapid method to detect the pesticide residues is presented by using Au nanoparticles/dragonfly wing (AuNPs/DW) arrays as SERS-active substrate. The AuNPs/DW substrate is prepared by decorating the AuNPs on the DW surface with a simple two-step method, meanwhile, the microstructure properties and SERS signal are characterized by the scanning electron microscopy (SEM) and confocal microprobe Raman system. The experimental results show that compared with the conventional Raman spectroscopy, the AuNPs/DW substrate can enhance the Raman signal dramatically due to the largescale nanosized protrusions on the DW surface. The detection limit for rhodamine 6G (R6G) could reach as low as 10−8 M, which is important for the analysis of the most components and structures of samples. For the application of the three-dimensional AuNPs/DW, the micro-sample of thiram and carbaryl can also be detected quantitatively, and the detection limit both reach up to 10−7 M. The above phenomenon indicate that three-dimensional nanostructure AuNPs/DW is a promising SERS substrate for the test of samples in low concentration. Hence, our study will provide an effective approach for the rapid, sensitive and stable trace detection of organic molecular species.