A new, highly sensitive surface enhanced Raman scattering (SERS)-based immunoassay platform was prepared using silver nanoparticle (AgNP)-decorated electrospun fibers as the capture substrate. We used electrospinning and silver mirror reaction to generate AgNP-decorated polycaprolactone (PCL) fiber matrix (Ag-PCL). The resultant capture substrates obtained were bi-directionally porous, free-standing, and flexible. AgNP formation on the PCL fibers was confirmed via SEM, AFM, XPS, and TGA analysis. In addition, gold nanoparticles immobilized with a Raman reporter, 4-mercaptobenzoic acid (4-MBA), were prepared as the SERS tag. This tag could significantly enhance the SERS signal via generation of additional hot spots between AgNPs on fibers and AuNPs. For a model immunoassay to detect prostate specific antigen (PSA), PSA antibodies were immobilized on both Ag-PCL and AuNP SERS tags. The large surface area of fiber substrates allowed the immobilization of large amounts of antibodies and their porous structures facilitated the assessment of the target antigen to immobilized antibodies. Binding of PSA between antibodies on AgNPs and AuNPs led to formation of a sandwich structure by the two metal nanostructures, and consequently, highly sensitive detection of PSA was possible up to a detection limit of 1 pg/mL within 1 h of reaction time. The developed SERS-based immunoassay platform produced uniform and reproducible SERS signals over the entire substrate area and from different samples.