For electrochemical carbon dioxide reduction (CO₂RR), CO₂-to-CO conversion is considered an ideal route towards carbon neutrality for practical applications. Gold (Au) is known as a promising catalyst with high selectivity for CO; however, it suffers from high cost and low mass-specific activity. In this study, we design and prepare a catalyst featuring uniform S-doped Au nanoparticles on N-doped carbon support (denoted as S-Au/NC) by an in situ synthesis strategy using biomolecules. The S-Au/NC displays high activity and selectivity for CO in CO₂RR with a Au loading as low as 0.4 wt.%. The Faradaic efficiency of CO (FE_CO) for S-Au/NC is above 95% at −0.75 V (vs. RHE); by contrast, the FE_CO of Au/NC (without S) is only 58%. The Tafel slope is 77.4 mV·dec⁻¹, revealing a favorable kinetics process. Furthermore, S-Au/NC exhibits an excellent long-term stability for CO₂RR. Density functional theory (DFT) calculations reveal that the S dopant can boost the activity by reducing the free energy change of the potential-limiting step (formation of the *COOH intermediate). This work not only demonstrates a model catalyst featuring significantly reduced use of noble metals, but also establishes an in situ synthesis strategy for preparing high-performance catalysts.