In order to improve the detection performance of surface-enhanced Raman scattering (SERS), a low-cost Au@Ag nanorods (Au@Ag NRs) substrate with a good SERS enhancement effect was developed and applied to the detection of malachite green (MG) in aquaculture water and crayfish. By comparing the SERS signal enhancement effect of five kinds of Au@Ag NRs substrates with different silver layer thickness on 4-mercaptobenzoic acid (4-MBA) solution, it was found that the substrate prepared with 100 µL AgNO3 had the smallest aspect ratio (3.27) and the thickest Ag layer (4.1 nm). However, it showed a good signal enhancement effect, and achieved a detection of 4-MBA as low as 1 × 10−11 M, which was 8.7 times higher than that of the AuNRs substrate. In addition, the Au@Ag NRs substrate developed in this study was used for SRES detection of MG in crayfish; its detection limit was 1.58 × 10−9 M. The developed Au@Ag NRs sensor had the advantages of stable SERS signal, uniform size and low cost, which provided a new tool for SERS signal enhancement and highly sensitive SERS detection method development.