A novel surface-enhanced Raman scattering (SERS)-based analytical technique was proposed to simultaneously detect two highly pathogenic bacteria, namely, Staphylococcus aureus (S. aureus) and Listeria monocytogenes (L. mono) by using a dual-recognition pattern with wheat germ agglutinin (WGA) and nucleic acid aptamers. WGA was modified onto Fe3O4@Au magnetic nanoparticles (MNPs) for the efficient capture of S. aureus and L. mono in complex samples (orange juice, extracts of lettuce, and human urine) within 15 min. The streptavidin (SA)/aptamers co-functionalized SERS tags were fabricated by covalent attaching two different Raman reporters and SA molecules onto 45 nm Au NPs and then conjugated with two biotin-aptamers that specifically bind to their target bacteria with high affinity and stability. The combined use of high-sensitive SERS tags, WGA-mediated magnetic enrichment, and SA-mediated aptamer conjugation remarkably improved the assay sensitivity. Under optimized conditions, the developed SERS biosensor can simultaneously detect the two target bacteria with high detection sensitivity (<6 cells/mL), favorable linear relation (10-107 cells/mL), and high accuracy (recovery rate <7.03%). Therefore, the proposed SERS platform is rapid, sensitive, easy to use, and thus show potential as a tool for the timely identification of pathogenic bacteria in real samples. Display omitted •A rapid and ultrasensitive SERS platform for S. aureus and L. mono detection was reported.•WGA modified-Fe3O4@Au MNP was proposed for broad-spectrum capture of multiple bacteria.•The SA/aptamer co-functionalized SERS tags exhibited higher affinity and stability.•The LODs for S. aureus and L. mono were 3 and 5 cells/mL, respectively.