The integration of novel surface-enhanced Raman scattering (SERS) nanoprobes and a microfl uidic dielectrophoresis (DEP) device is developed for rapid on-line SERS detection of Salmonella enterica serotype Choleraesuis and Neisseria lactamica . The SERS nanoprobes are prepared by immobilization of specifi c antibody onto the surface of nanoaggregate-embedded beads (NAEBs), which are silica-coated, dye-induced aggregates of a small number of gold nanoparticles (AuNPs). Each NAEB gives highly enhanced Raman signals owing to the presence of well-defi ned plasmonic hot spots at junctions between AuNPs. Herein, the on-line SERS detection and accurate identifi cation of suspended bacteria with a detection capability down to a single bacterium has been realized by the NAEB?DEP?Raman spectroscopy biosensing strategy. The practical detection limit with a measurement time of 10 min is estimated to be 70 CFU mL ?1 . In comparison with whole-cell enzyme-linked immunosorbent assay (ELISA), the SERS-nanoprobe-based biosensing method provides advantages of higher sensitivity and requiring lower amount of antibody in the assay (100-fold less). The total assay time including sample pretreatment is less than 2 h. Hence, this sensing strategy is promising for faster and effective on-line multiplex detection of single pathogenic bacterium by using different bioconjugated SERS nanoprobes.