Display omitted •A cauliflower-like super-hydrophobic SERS substrate by two-step laser processing.•The enhancement factor of the SERS substrate reaches 5.9 × 109 for R6G.•The detection limit of the SERS substrates for acetamiprid is as low as 10-5 g/L. Residues of organic pesticides continue to accumulate in the human body through the food chain, posing a serious threat to health. Therefore, the efficient and sensitive detection of pesticide residues is crucial. Surface-enhanced Raman scattering (SERS), as a powerful spectroscopic technique, holds great potential for the trace detection of pesticides. In this study, a cauliflower-like SERS substrate fabricated via a two-step laser processing is proposed for highly sensitive detection of acetamiprid. The cauliflower-like structure features a large surface area, providing numerous hotspots and abundant adsorption sites for the target molecules. Furthermore, the rich micro/nano structures on the surface confer super-hydrophobicity properties to the substrate, effectively enriching the analyte and greatly enhancing the SERS performance. As a result, the cauliflower-like super-hydrophobic substrate demonstrates a detection limit as 10-15 M for the typical dye analyte (R6G), with an enhancement factor of 2.6 × 108. Additionally, the substrate exhibits excellent signal reproducibility with a relative standard deviation of 8.33 %. In practical pesticide detection, the detection limit for acetamiprid on orange is 10-4 g/L, significantly surpassing the national standard. In conclusion, the cauliflower-like super-hydrophobic substrate enhances the sensitivity, uniformity, reproducibility, and reliability of SERS signals in detection process, showing tremendous potential in the field of food safety.