This study aims to develop a simple, sensitive, low-cost, environmentally friendly and flexible surface-enhanced Raman scattering (SERS) platform, combined with a portable Raman spectrometer, for the rapid and on-site SERS detection of bacteria. Commercial tobacco packaging paper (TPP) with little background interference was used as a loading medium that effectively adsorbed Au nanoparticles and provided sufficient “hot spots”. This Au-tobacco packaging paper (Au-TPP) substrate used as a flexible SERS platform can maximize sample collection by wiping irregular surfaces, and was successfully applied to the on-site and rapid detection of pathogenic bacteria. Raman fingerprints of pathogenic bacteria can be obtained by SERS detection of spiked pork using wipeable Au-TPP, which verifies its value in practical applications. The results collected by SERS were further verified by polymerase chain reaction (PCR) results. It showed several advantages in on-site SERS detection, including accurate discrimination, simple preparation, easy operation, good sensitivity, accuracy and reproducibility. This study indicates that the established flexible SERS platform has good practical applications in pathogenic bacterial identification and other rapid detections.