Traumatic brain injury (TBI) is a sudden brain injury due to an external force that causes a large number of deaths and permanent disabilities every year. S100B has been recognized as a potential objective quantitative biomarker for screening the prognosis of TBI and severe head injury. In this article, an anti-S100B monoclonal antibody was immobilized on cysteamine (Cy) functionalized gold nanoparticles (AuNPs) by EDC-NHS chemistry, which enabled S100B resonance Rayleigh scattering (RRS) detection based on antibody-labeled gold nanoparticles. The prepared conjugates were characterized by ultraviolet-visible spectroscopy (UV-vis), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Based on the specific binding of the antibody and antigen, the RRS intensities at 381 nm and 541 nm wavelengths were significantly enhanced, and thus a dual wavelength overlapping resonance Rayleigh scattering (DWO-RRS) method was established. The scattering intensity of the two overlapping peaks was proportional to the concentration of S100B in the range of 0.05-4.5 ng mL-1 with a detection limit of 0.002 ng mL-1. The proposed DWO-RRS method is time-saving, simple, sensitive, and can be used to determine the concentration of S100B in human serum with satisfactory results, which has a promising application in the early diagnosis of TBI.