The integration of Raman-labeled nanoaggregate-embedded beads (NAEBs) for high performance SERS analysis of single mimic pathogen on a self-designed dielectrophoretic chip is demonstrated. The Raman tags called NAEBs are silica-coated, dye-induced aggregates of a small number of gold nanoparticles (AuNPs). In this work, NAEBs consisting of a Raman dye tetramethyl-rhodamine-5- isothiosyanate (TRITC) are chemically functionalized with streptavidin to detect biotin-functionalized polystyrene (PS) microspheres which mimic as pathogens. The sample solution of completely mixed streptavidin-functionalized NAEBs and biotin-functionalized PS microspheres is pumped into the microfluidic channel of a dielectrophoretic chip. By giving an AC voltage on the embedded electrodes, a single mimic pathogen can be caught via the non-contact dielectrophoretic force and suspended at the central cross of four aluminum electrodes for subsequent Raman spectroscopic detection. The SERS signal of TRITC is used as a spectral signature of specific mimic pathogen recognition, otherwise only the background Raman signal of a PS microsphere is observed. A pathogen-specific biosensor based on the dielectrophoresis-Raman spectroscopy system is developed, and the proof-ofconcept is confirmed by the specific molecular interaction model of streptavidin with biotin. Therefore, the on-chip multiplex SERS analysis of pathogens can be anticipated by employing different dye-tagged NAEBs simultaneously in a sample solution. We believe this bioassay has the ability to screen and detect multiple pathogens with minimal sample processing and handling even a small number of pathogens is present.