Carbon dioxide (CO₂) is the primary greenhouse gas that has emerged as a serious threat to environmental conditions and public health. Many studies have shown that cities are the areas with the highest CO₂ emissions. It is of great significance to comprehensively analyze the impact of urban spatial structure on CO₂ emissions so that CO₂ emission can be reduced through reasonable urban planning. Previous studies have mainly focused on the relationship between urban two-dimensional spatial structure and CO₂ emissions, indicating that urban expansion is the main reason for the sharp increase in CO₂ emissions. Although the urban three-dimensional spatial structure also significantly affects CO₂ emissions, the relationship between them still lacks in-depth investigations. Therefore, the purpose of this research is to explore the relationship between three-dimensional spatial structure and CO₂ emissions and to reveal the associated scale effect at multiple grid scales by taking Guangzhou as an example. These tasks were conducted by using the Pearson correlation test, random forest regression, and scale analysis. The results indicated that:(1) The effect of three-dimensional spatial structure on CO₂ emissions is greater than that of two-dimensional land cover. Among them, (high-rise) building density, building coverage ratio, floor area ratio, and population density were the key factors affecting CO₂ emissions, which could increase energy consumption and CO₂ emissions by directly increasing human activities or intensifying urban heat island effects and the associated energy consumption. (2) Scale effect is obvious in terms of the relationship between three-dimensional spatial structure and CO₂ emissions. Although the three-dimensional spatial structure is the major influencing factor at all scales, the order of importance of influencing factors varies among different scales. The dominance of high-rise buildings is stronger in small and large grid scales, and the dominance of the overall layout of buildings is stronger in moderate grid scales. Therefore, determining a reasonable analysis scale is another important aspect of revealing the influencing mechanisms of CO₂ emissions. (3) As a representative of modern compact cities, Guangzhou should not unilaterally pursue the compact layout of the urban three-dimensional space, which is not beneficial to the development of low-carbon city. Rational planning of the urban three-dimensional spatial structure is an important approach to low-carbon city construction. In conclusion, it is necessary for policy-makers to pay attention to the reasonable layout of the three-dimensional spatial structure at macro scale and the difference in the impact of three-dimensional spatial structure at different scales. Additionally, policy-makers should attach importance to the reasonable development of urban fringe areas. Furthermore, policy-makers should reduce the compact degree of urban central buildings and develop a multi-center urban development pattern, which can effectively reduce the CO₂ emissions and promote the sustainable development of low-carbon cities. In summary, this research can support the rational optimization of urban three-dimensional spatial structure, and can help to achieve the carbon peaking and carbon neutrality goals of China.