In this study, silver nanoparticles (Ag NPs) were decorated on polyvinyl alcohol/chitosan nanofibers (Ag@PVA/PEI) as a SERS substrate for norfloxacin detection by electrospinning and in-situ reduction. Silver ions were anchored on the surface of PVA/CS nanofibers by free amino groups in chitosan framework, and then reduced by ascorbic acid to form AgNPs, which were spherical and uniformly decorated on the surface of PVA/CS membranes. The SERS performance of the prepared substrate was evaluated by using rhodamine 6G (R6G) as a probe. The detection limit was 10−6 M. In addition, the Ag@PVA/CS substrate can be used for the identification and detection of norfloxacin. The detection limit was 10-5 M. The exceptional SERS performance, sensitive, flexibility, low cost, render the obtained SERS substrate in this study a promising product for detection in practical applications.