The receptor-binding domain of the SARS-CoV-2 spike mediates the key to binding the virus to the host receptor, but capturing the molecular signal of this spike RBD remains a formidable challenge. Here, we report a new surface-enhanced Raman spectroscopy (SERS) approach, which used gold nanoparticles prepared by low-speed constant-temperature centrifugation by bromine and calcium ions in two cleaning steps as the enhanced substrate to rapidly and accurately detect spike RBD large protein molecules in body fluids. The detection signal was extremely stable, and the orientation of the spike RBD on the enhanced substrate surface was also determined. This approach was specific in distinguishing different SARS-CoV-2 variants of spike RBD, including Delta, Beta, Gamma, and Omicron. Additionally, the enhanced substrate can identify biologically active or inactive spike RBD. This two-step cleaning enhanced substrate opens up opportunities not only for early diagnostics of SARS-CoV-2 virus but also for developing targeted drugs against viruses.