Surface-enhanced Raman spectroscopy (SERS) is a promising analytical tool, but simultaneous detection of multiple targets using SERS remains a challenge. Herein, a cauliflower-inspired 3D SERS substrate with intense hot spots was prepared through sputtering Au nanoparticles (Au NPs) on the surface of polydimethylsiloxane coated anodic aluminum oxide (PDMS@AAO) complex substrate. As a result, the cauliflower-inspired 3D SERS substrate achieved the highest SERS activities at a sputtering time of 8 min. Under the optimal conditions, this SERS substrate possessed a low detection limit of 10-12 M, excellent enhancement uniformity (relative standard deviation, RSD = 4.57%) and high enhancement factor (2.2 × 106) for 4-mercaptobenzoic acid (4-MBA). Furthermore, the results of Raman showed that the 3D-Nanocauliflower SERS substrates could realize the simultaneous label-free detection for three mycotoxins (aflatoxin B1, deoxynivalenol, and zearalenone) in maize for the first time. It behaved good linear relationship between the concentrations and Raman intensities of aflatoxin B1, zearalenone, and deoxynivalenol. For the three mycotoxins, this method exhibited the limit of detection (LOD) of 1.8, 47.7, and 24.8 ng/mL (S/N = 3), respectively. The 3D-Nanocauliflower SERS substrates with dense hot spots presented remarkable SERS effect and activity, which could be act as a potential candidate for SERS substrate applied in the rapid and label-free detection.