Surface-enhanced Raman scattering (SERS) spectroscopy, a technique with high sensitivity and high resolution, can provide the fingerprint vibration spectrum of the analyzed molecule and realize the nondestructive measurement of the sample, which has very important applications in field of spectrum analysis. The sensitivity, stability, and reproducibility of SERS spectrum depend on the performance of SERS substrate. The common SERS rigid substrates have the defects of complex preparation process, poor flexibility, inconvenience to carry, and fragility. Paper-based SERS substrate, which uses paper as support material, can effectively overcome the shortcomings of rigid substrates and meet the commands of fast, handy, and personized analysis and measurement in the future. In this review, we summarize the main methods (direct dipping/dropping, inkjet printing, chemical reaction, and physical spray coating) for the preparation of paper-based SERS substrates, and discuss the applications in biomedical analysis and sensing, environmental monitoring, and food safety.