A novel method based on the separation and enrichment effect of magnetic beads and the fully complementary hybridization of two DNA strands was developed for highly sensitive detection of bacterial DNA using a surface-enhanced Raman spectroscopy (SERS) with 5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB)-modified gold nanoparticles as reporter probes. Capture probe was immobilized onto the surface of streptavidin-enwrapped magnetic beads ( SA-MB ) through high affinity between biotin and avidin, by which the target bacterial DNA sequences that connected with the reported probe decorated AuNPs with DTNB and SH-DNA ( AuNPs@DTNB@DNA) were captured and loaded onto the magnetic beads by the hybridization reaction with the capture probe. Compared with previous methods, this design shortened the distance between particles by the ways that the magnetic beads tempted to nanoparticles aggregation, and produced the plasma resonance coupling effect, which increased the SERS signal significantly. The results showed that, under the optimized conditions and in the concentration range from 5 pmol/L to 5 nmol/L, the method performed a good linear relationship between Raman intensity and DNA concentration. The limit of detection ( LOD) of bacterial DNA was estimated to be 5 pmol/L. The method is simple and low cost, and can be used in the sensitive and selective detection of bacterial DNA.