Although single-particle cryogenic electron microscopy(cryo-EM)has been applied extensively for elu-cidating many crucial biological mechanisms at the molecular level,this technique still faces critical challenges,the major one of which is to prepare the high-quality cryo-EM specimen.Aiming to achieve a more reproducible and efficient cryo-EM specimen preparation,novel supporting films including graphene-based two-dimensional materials have been explored in recent years.Here we report a ro-bust and simple method to fabricate EM grids coated with single-or few-layer reduced graphene oxide(RGO)membrane in large batch for high-resolution cryo-EM structural determination.The RGO mem-brane has decreased interlayer space and enhanced electrical conductivity in comparison to regular graphene oxide(GO)membrane.Moreover,we found that the RGO supporting film exhibited nice particle-absorption ability,thus avoiding the air-water interface problem.More importantly,we found that the RGO supporting film is particularly useful in cryo-EM reconstruction of sub-100-kDa bio-molecules at near-atomic resolution,as exemplified by the study of RBD-ACE2 complex and other small protein molecules.We envision that the RGO membranes can be used as a robust graphene-based sup-porting film in cryo-EM specimen preparation.