Silver nanoparticle (AgNP)/poly(N-isopropylacrylamide) (PNIPAAm) composite solution was electrospun followed by crosslinking to fabricate AgNP/PNIPAAm composite nanofiber mats. Embedded AgNPs were found to align along the PNIPAAm nanofibers, which generate more hotspots upon laser-induced plasmonic heating. This kind of AgNP/PNIPAAm mats show enhanced surface-enhanced Raman scattering (SERS) effect upon continuous pulsed laser irradiation, showing a laser heating sensitive' SERS effect. Laser-induced plasmonic heating can also tune the temperature-responsive interaction between PNIPAAm and analytes and has been applied in the on-site laser heating sensitive' separation and SERS detection of analytes. The composite nanofiber mats also showed good reproducibility and stability. What is more, this laser heating sensitivity' has also been applied in the direct SERS detection of adenosine in urea solution without urea interference. Together with the advantages of ease of large-scale fabrication, stable and laser heating sensitivity', AgNP/PNIPAAm mats might find application in sensitive multicomponent biosensing. Copyright (c) 2016 John Wiley & Sons, Ltd.