Developing efficient carbon-based metal-free electrocatalysts can bridge the gap between laboratory studies and practical applications of CO₂ reduction. However, along with the ambiguous understanding of the active sites in carbon-based electrocatalysts, carbon-based electrocatalysts with high selectivity and satisfactory stability for electroreduction of CO₂ remain rare. Here, using the nitrogen rich silk cocoon as a precursor, carbon-based electrocatalysts with intrinsic defects can be prepared for efficient and long-term electroreduction of CO₂ by a simple two-step carbonization. The obtained electrocatalyst can catalyze CO₂ reduction to CO with a Faradaic efficiency of ~ 89% and maintain good selectivity for about 10 days. Particularly, our experimental studies suggest that in-plane defects are the main active sites on which the rate-determining step for CO₂ reduction should be the direct electron transfer to CO₂ but not the proton-coupled electron transfer. Further theoretical calculations consistently demonstrate that the intrinsic defects in carbon matrix, particularly the pentagon-containing defects, act as main active sites to accelerate the direct electron transfer for CO₂ reduction. In addition, our synthetic approach can convert egg white into efficient catalysts for CO₂ electroreduction. These findings, providing new insights into the biomass-derived catalysts, should pave the way for fabricating efficient and stable carbon-based electrocatalysts with catalytically active defects by using naturally abundant precursors.