The invention relates to the technical field of trace molecule detection, in particular to preparation and application of Raman enhancement substrates based on multi-layer composite structures. The preparation method comprises the following steps that multiple substrates are placed on a
quartz baseplate, an insulation part and a
molybdenum sheet are placed on the upper surface of each substrate insequence, and the substrates are placed in pre-heated heating equipment for a
thermal reaction; the substrates are taken out and cooled, the insulation parts are removed, and
molybdenum oxide substrates are prepared; a SnCl2 solution with the concentration of 10 nmol / L is prepared, and the pH value of the SnCl2 solution is adjusted to 5-6; the prepared
molybdenum oxide substrates are placed in the SnCl2 solution, placed in a pre-heated heating table for Sn
ion interlayer intercalation, taken out and blow-dried through a
nitrogen gun, and the Raman enhancement substrates based on the multi-layer composite structures are prepared. The preparation method is easy and quick to implement, an intralayer structure of
molybdenum oxide is not changed, by introducing interlayer ions,
doping is achieved, and the prepared substrates are stable in quality and high in sensitivity.