A method of fabricating surface-enhanced Raman scattering (SERS) substrate by one-step exposure of mixed electron beam resists is reported. Stable and scattered nanospheres were produced by mixing immiscible hydrogen silsesquioxane (HSQ) and polymethyl methacrylate (PMMA) electron beam resists in equal quantity, followed by electron beam exposure with an appropriate dosage of 2000 μC/cm² and development. Thin Au film was then deposited using electron beam evaporation to form SERS substrates with superior Raman enhancement effects. The size distribution of the microspheres with the relative standard deviation of 7.56% is uniform, and the gaps between rough Au layers and the nanospheres could provide abundant SERS "hot spots". The SERS substrates thus fabricated showed superior performance for different targets. A SERS enhancement factor of 5.8×10⁶ and detection limit of 1.06×10^-8 mol/L for 4-mercaptophenylboronic acid (4-MPBA), and the detection limits of 7.08×10^-9 mol/L for rhodamine 6G (R6G) and 7.94×10^-10 mol/L for melamine in water were achieved. For melamine, a wide detection range (1.0×10^-9-1.0×10^-5 mol/L) with a good linearity (R²=0.952) was achieved. The pronounced performance of detecting melamine makes the SERS substrates very overwhelming among the ones reported. The facile and repeatable approach of preparing high-performance SERS substrates by exposing the mixture of different resists proposed here is undoubtedly favorable for the exploration of novel SERS substrates and their fabrication.