SERS primers have been used to directly detect specific PCR products utilizing the difference in adsorption of single-stranded and double-stranded DNA onto nanoparticle surfaces. Herein, seven parameters important for improved positive SERS assays for real applications were investigated. First, we applied a model system for optimization experiments, followed by a PCR assay to detect pathogen DNA, and then the introduction of a new assay that utilizes the 5'→3' exonuclease activity of Taq DNA polymerase to partly digest the SERS probe, generating dye-labeled single-stranded DNA increasing the SERS signals for detection of pathogen DNA. Applying the model system, it was found that uni-molecular SERS primers perform better than bi-molecular SERS primers. However, within the PCR assays, it was found that uni- and bi-molecular SERS primers performed very similarly, and the most reproducible results were obtained using the 5'→3' exonuclease digestion assay. These SERS-based assays offer new routes over conventional fluorescence-based techniques without compromising sensitivity or selectivity.