Surface-enhanced Raman spectroscopy (SERS) is a useful analytical technique for detecting extremely small amounts of molecules. Herein, we designed a paper-based quasi-three-dimensional SERS substrate (P-SERS) that can provide potential to improve Raman analyses for food safety, pesticide poisoning, precision medicine, drug abuse and DNA/RNA testing. The sensitive, low-cost, flexible and disposable SERS substrate could be easily fabricated by physical deposition of gold nanoparticles array onto a filter paper. In this case, we are able to create non-continuous Au islands on the fiber surfaces, where the gaps between AuNPs can dramatically generate the high electric field to enhance Raman signal of target molecules.