An aptamer-based sensitive method was developed here for detection of multiple foodborne pathogens in food matrix by surface-enhanced Raman scattering (SERS) technology. Polydimethylsiloxane (PDMS) film was first prepared and then coated with gold nanoparticles (AuNP) to act as an active substrate for the enhancement of Raman scattering. The as-prepared Au-PDMS film was functionalized with specific pathogen aptamers (Apt) to capture the targets. In addition, aptamers functionalized AuNP integrated with Raman reporters (4-Mercaptobenzoic acid (4-MBA)/Nile blue A (NBA)) were fabricated as pathogen-specific SERS probes. In this scheme, pathogens were first captured by Apt-Au-PDMS film and then bind with SERS probes to allow the formation of a sandwich assay to complete the sensor module for the detection of multiple pathogens. With Vibrio parahaemolyticus and Salmonella typhimurium as model targets, this protocol can selectively detect 18 cfu/mL and 27 cfu/mL, respectively. Furthermore, this platform can be successfully applied to detect pathogens in seafood samples with recoveries ranging from 82.9% to 95.1%.