We demonstrate a novel approach for fabricating surface enhanced Raman scattering (SERS) substrates for single bacterial biosensing based on Ag cylindrical nanotrough networks (CNNs). This approach is developed with large scalability by leveraging a cellulose nanofiber template fabrication via facile electrospinning. Specifically, a concave nanotrough structure consisting of interconnected concave Ag nanoshells is demonstrated by depositing a thin layer of Ag atop a sacrificial electrospun nanofiber template and then completely removing the cellulose core in water. Our investigations of the scattering properties and SERS performances of single isolated Ag nanotroughs of different diameters reveal that nanotrough-based substrates provide tunable optical responses and enhanced SERS intensities. Further, Ag CNNs are fabricated in highly interconnected networks that yield reproducible SERS signals for molecular monolayers and whole bacterial cells, enabling rapid spectral discrimination between different bacterial strains. Finally, by performing principal component analysis on a large number of measured SERS spectra (40 spectra per bacterium), we demonstrate successful spectral discrimination between two types of Escherichia coli (E. coli) bacteria, that is, E. coli K12 with its derivative E. coli DH 5 alpha and E. coli BL21(DE3). The demonstrated cost-effective substrates feature several advantages over conventional SERS substrates including environmentally friendly and scalable fabrication compatible with versatile devices and provide an alternative approach to rapid SERS detection and screening of biochemicals.