Surface-enhanced Raman spectroscopy (SERS) has shown its ability to characterize a biological substance down to a single-molecule level without a specific biorecognition mechanism. Various nanofabrication technologies enable SERS substrate prototyping and mass manufacturing. In this study, SERS substrate simulation, fabrication, and prototyping based on two-photon polymerization (2PP) are discussed. The developed substrates showed up to 10^6 Raman signal enhancements, comparable to those of commercial substrates. Moreover, the rapid prototyping of SERS substrates based on 2PP takes less than a minute to 2 hours, depending on the fabrication approach and aspect ratio requirements. The process is well-controlled and reproducible for achieving a uniform distribution of nanostructure arrays. The developed SERS substrates can be used for a broad range of applications and the characterization of different molecules.