We report a novel approach for fabricating active surface-enhanced Raman scattering (SERS) substrate for sensitive detection. This approach is based on the assembling of gold nanoparticles (AuNPs) onto the electrospun polycaprolactone (PCL) nanofiber film. The hydrophobic surface of PCL nanofiber film was pretreated using UV-inducing graft polymerization with acrylic acid. Afterwards this PCL nanofiber film was incubated with the AuNP solution to promote the assembly of AuNPs onto the PCL nanofibers and the formation of SERS active substrate. 4-aminothiophenol (4-ATP) molecule was used as a test probe for SERS experiments, indicating that the substrate has high sensitivity to SERS response. Our method has great advantage in term of environment-friendly synthesis, large-scale, high stability and good reproducibility. This highly active SERS substrate can be employed to detect the drug molecule, 2-thiouracil.