The fabrication of surface enhanced Raman spectroscopy (SERS) substrates with controlled high density hot spots still remains challenging. Herein, we report highly effective SERS substrates containing the self-generating (SG) nanogaps from polystyrene nanosphere monolayer through isotropic plasma etching. The emergence of multimode hot spots, i.e., metal film over nanosphere (MFON)-like hot spots (closed gaps, 0 nm), individual self-aligned hot spots (discrete gaps, > 20 nm) and three-dimensional (3D) hot spots (nanogaps, 1–10 nm), makes the SG SERS substrates superior as compared to the traditional MFON or the well-ordered self-aligned SERS substrates in terms of enhancement, uniformity, and reproducibility. The SG SERS substrates can function as the excellent SERS platforms for trace molecule detection in the practical application fields.