In this study, a surface enhanced Raman scattering (SERS) sensor based on Au@Ag NPs solid-phase substrate combined with chemometrics was constructed for the discrimination of three pathogenic bacteria (Staphylococcus aureus, Escherichia coli and Listeria monocytogenes). The Au@Ag NPs were synthesized and self-assembled on filter paper using the dip-coating method. The good absorbency of the filter paper immobilized the bacteria on the substrate, increased the interaction between the bacteria and the substrate, and enhanced the SERS signal of the bacteria. The main peaks of the bacterial spectra were close to each other, but the relative intensities of the vibrational peaks were significantly different, and each strain exhibited unique Raman peaks. The combination of partial least squared discriminant analysis (PLS-DA) method with bacterial SERS allowed the effective identification of the three bacteria. Moreover, the method was applied for the quantitative detection of Staphylococcus aureus with a minimum detection concentration of 10 cfu/mL.