The identification and timely detection of pathogenic bacteria is critical to ensuring safe food, health, and water. Although surface enhanced Raman scattering (SERS) methods have been used for pathogen characterization and single molecule sensing, the challenge of detecting pathogens in very low numbers using an optimal substrate that is sensitive and reproducible is still a challenge. In this report, we have developed and explored a novel SERS active substrate of 60-80 nm diameter through the assembly of Ag nanocrystals (AgNCs) into Ag nanospheres (AgNSs). A finite difference time domain (FDTD) analysis of the electromagnetic field produced by these structures and the enhancement factor calculations indicated that an enhancement of 108 was possible using the 633 or 785 nm excitation. The exact enhancement factors (EF) through the experimental results were calculated to be 2.47 × 107, which is close to that obtained through the FDTD analysis. Preliminary characterization of the SERS substrate was demonstrated using various labels from the fluorescent dye and nonfluorescent small molecules. More importantly, these novel SERS active substrates when used for pathogenic bacteria detection could detect cells as few as 10 colony forming units/mL (CFU/mL). Using canonical variate analysis (CVA) in conjunction with Raman spectra, differentiation of three key pathogens (E. coli O157, S. typhimurium, and S. aureus) including live and dead cells was also accomplished. With further optimization of the SERS substrate, single cell detection is possible.