The invention discloses preparation of an SERS (Surface Enhanced Raman Scattering) substrate and application of the SERS substrate to cancer detection and belongs to the field of nanomaterials. The SERS substrate is TiO2 inverse opal plated with 80nm Au, and the bore diameter of the TiO2 inverse opal is 290nm. In the SERS substrate, the bore diameter of the TiO2 inverse opal is matched with the diameter (about 90nm) of an exosome of a to-be-detected object, and the exosome can make full contact with the SERS substrate; and on the other hand, the TiO2 inverse opal with the bore diameter being 290nm can generate a slow light effect during analyte detection under the excitation of a 633nm laser, and then an SERS signal of the exosome itself can be better obtained. By use of the SERS substrate, the phosphorylation degree of exosome protein extracted from plasma can be quickly analyzed, and then a cancer is diagnosed; consumed time is short, and sensitivity is high; and a new thought is provided for quick diagnosis of a non-invasive in-vitro cancer.