Development of a rapid, sensitive, and multiplex pathogen DNA sensor enables early diagnosis and, subsequently, the proper treatment of infectious diseases, increasing the possibility to save the lives of infected patients. Here, the development of an ultrasensitive and multiplex pathogen DNA detection method that combines a patterned Au nanowire (NW)-on-film surface-enhanced resonance Raman scattering (SERRS) sensor with an exonuclease III-assisted target DNA recycling reaction is reported. Multiple probe DNAs are added to the target DNA solution, and among them, only the complementary probe DNA is selectively digested by exonuclease III, resulting in the decrease in its concentration. The digestion process is repeated by recycling of target DNAs. The decrease of the complementary probe DNA concentration is detected by SERRS. Combining the high sensitivity of the NW-on-film sensor and the target recycling reaction significantly improves DNA detection performance, resulting in the detection limit of 100 fM corresponding to 3 amole. By positioning Au NWs at specific addresses, multiple pathogen DNAs can be identified in a single step. Clinical sample tests with multiple genomic DNAs of pathogens show the potential of this sensor for practical diagnosis of infectious diseases.