Anew and facile way to synthesize a free-standing and flexible surface-enhanced Raman scattering (SERS) substrate has been successfully developed, where high SERS-active Ag dimers or aligned aggregates are assembled within poly(vinyl alcohol) (PVA) nanofibers with chain-like arrays via electrospinning technique. The aggregation state of the obtained Ag nanoparticle dimers or larger, which are formed in a concentrated PVA solution, makes a significant contribution to the high sensitivity of SERS to 4-mercaptobenzoic acid (4-MBA) molecules with an enhancement factor (EF) of 10(9). The superiority of enhancement ability of this Ag/PVA nanofiber mat is also shown in the comparison to other substrates. Furthermore, the Ag/PVA nanofiber mat would keep a good reproducibility under a low concentration of 4-MBA molecule (10(-6) M) detection with the average RSD values of the major Raman peak less than 0.07. The temporal stability of the substrate has also been demonstrated. This disposable, easy handled, flexible free-standing substrate integrated the advantages including the superiority of high sensitivity, reproducibility, stability, large-scale, and low-cost production compared with other conventional SERS substrates, implying that it is a perfect choice for practical SERS detection application.