Gold nanorods (AuNRs) can be successfully co-assembled with Ag nanowires (AgNWs) to form a kind of AuNR-AgNW nanocomposite by electrostatic attraction, in which the AuNRs are arranged along the long axial direction of the AgNWs with a preferential string-like alignment. The assembled AuNR-AgNW nanocomposites are then further embedded within polyvinyl alcohol (PVA) nanofibers by electrospinning, by which both AuNRs and AgNWs can be stabilized and arranged along the axis of polymer nanofibers. When the polymer nanofibers are aligned by collecting on a copper mesh with a woven structure, the AuNR-AgNW nanocomposites assembled within the electrospun nanofibers are also arranged. The influences of the AuNR-AgNW assemblies with different amounts of AuNRs attached on AgNWs on the optical properties and surface enhanced Raman scattering (SERS) enhancement have been investigated. The resulting AuNR-AgNW/PVA electrospun mats show red-shifted and broader absorption bands and higher SERS performances compared with the normal casting films with randomly dispersed AuNRs and AgNWs, or electrospun mats with monometallic components, due to the order alignment of AuNR-AgNW nanocomposites on a large scale.