This work presents the rapid detection of Bacillus subtilis spores, harmless simulants for Bacillus anthracis, using surface-enhanced Raman spectroscopy (SERS) on silver film over nanosphere (AgFON) substrates. Calcium dipicolinate (CaDPA), a biomarker for bacillus spores, can be extracted effectively from spores with nitric acid and successfully detected by SERS. The highly tunable nature of AgFON optical properties was exploited to establish general optimization conditions. AgFON surfaces optimized for 750-nm laser excitation have been characterized by UV-vis diffuse reflectance spectroscopy. The SERS signal from extracted CaDPA was evaluated over the spore concentration range 10-15-10-1212 M to determine the adsorption capacity of the AgFON surface and the limit of detection (LOD). These sensing capabilities have been successfully transitioned to an inexpensive, portable Raman spectrometer. Using the extraction method and this field-portable instrument, the anthrax infectious dose of 104 spores were detected with only a 5-second collection period on a one-month-old prefabricated AgFON substrate.