The invention discloses a preparation method of an SERS substrate of Au@Ag nanoparticles. According to the method, gold nanoparticles are prepared by reducing chloroauric acid with sodium citrate, thegold nanoparticles are modified with 4-sulfydryl aniline to link silver ions, and a certain proportion of ascorbic acid and sodium hydroxide are added to reduce a silver shell. Meanwhile, the form size of the core-shell structure is controllable, plasma resonance is realized through high coupling of the structure, and the so-called hotspots are formed in the core-shell structure, so that 4-sulfydryl aniline serving as an internal standard molecule has very high Raman signals. The Au@Ag core-shell structure is modified with 4-sulfydryl phenylboronic acid to capture glucose molecules, so that qualitative and quantitative detection of glucose is realized. The method has the advantages as follows: the internal standard substance is used for signal comparison, interference of complex substances in samples (such as urine) can be avoided, the detection sensitivity is improved, the preparation cost is low, and qualitative and quantitative detection of glucose is really realized.