Abstract Although surface-enhanced Raman spectroscopy (SERS) substrates have achieved high sensitivity, it is still difficult to apply these SERS protocols to the on-site detection of real samples due to the SERS substrate being fabrication-complexed, unstable, reproducible, or unable to be applied in batch production. Here, a large-scale ordered two-dimensional array of Au nano-hemispheres was developed through electron beam vaporization of Au onto the easy-available commercialized anodic aluminum oxide (AAO) template with two-layer nanostructures of different diameters. The uniform Au nano-hemisphere is reliable for SERS detection of malachite green (MG) due to the relative standard deviation (RSD) of the SERS intensities at different locations less than 10%. With the optimized excitation wavelength, solvent effect and pH environment, the linear range of MG detection spans from 10–10 to 10–6 mol/L with a limit of detection (LOD) of 4×10–10 mol/L. The enhancement factor can reach 1.2×106. After extraction with acetonitrile and dichloromethane, MG in the spiked tilapia was detected with satisfactory recovery. The results indicate that the Au nano-hemisphere array can be expected to greatly facilitate SERS practical applications in detecting harmful food additives and chemicals due to the advantages of chemical inertness, physical robustness, simple fabrication, controllability, large-area uniformity, and large-batch production.