In this report, a simple and highly selective homogeneous sandwich immunoassay was developed for ultrasensitive detection of Staphylococcal Enterotoxin B (SEB) using Surface Enhanced Raman Scattering (SERS). The assay uses polyclonal-antibody functionalized magnetic gold nanorod particles as capture probes for SEB, which can be collected via a simple magnet. After separating SEB from the sample matrix, they are sandwiched by using binding-specific antibody-antigen pairs with the help of gold nanorod particles. Gold nanorod particles are bifunctional by design and contain self-assembled monolayers (SAMs) of a SERS tag molecule (5,5-dithiobis (2-nitrobenzoic acid), DTNB) and carboxylic functionalities of DTNB for coupling with a suitable antibody. The correlation between the SEB concentration and SERS signal was found to be linear within the range of 3 fM to 0.3 mu M. The limit of detection for the assay was determined to be 768 mu M (ca., 9250 SEB molecules per 20 mu L sample volume). The gold heterogeneous assay system for SEB detection was also compared with the same SERS probes and gold-coated surfaces as capture substrates. The developed method was further evaluated for detecting SEB in artificially contaminated milk. Finally, the method was used for investigating the SEB specificity on bovine serum albumin (BSA) and avidin.