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With the development of cryo-electron microscopy (cryo-EM), high-resolution structures of macromolecules can be reconstructed by the single particle method efficiently. However, challenges may still persist during the specimen preparation stage. Specifically, proteins tend to adsorb at the air-water interface and exhibit a preferred orientation in vitreous ice. To overcome these challenges, we have explored dual-affinity graphene (DAG) modified with two different affinity ligands as a supporting material for cryo-EM sample preparation. The ligands can bind to distinct sites on the corresponding tagged particles, which in turn generates various orientation distributions of particles and prevents the adsorption of protein particles onto the air-water interface. As expected, the DAG exhibited high binding specificity and affinity to target macromolecules, resulting in more balanced particle Euler angular distributions compared to single functionalized graphene on two different protein cases, including the SARS -CoV-2 spike glycoprotein. We anticipate that the DAG grids will enable facile and efficient three-dimensional (3D) reconstruction for cryo-EM structural determination, providing a robust and general technique for future studies.