The graphene oxide (GO) membrane holds great promise in desalination and green energy fields due to its naturally occurring nanochannels, which provide significant advantages in gas and ion filtration. In this study, the sizes and distributions of nanopore/channels in GO membranes and the relationship of GO flakes size and membrane thickness are explored by molecular dynamics simulations. Our results demonstrate that the size of GO flakes influences the distribution of nanopore sizes in GO membranes, while it almost has no influence on the total nanopore area in membranes with the same thickness. Additionally, our findings confirm that the total nanopore area of the GO membranes decreases exponentially as the membrane thickness increases, which is consistent with experimental observations. To expand the range of nanopore size regulation, we developed a columnar-array substrate model to create GO membranes customized for specific filtration functions, such as virus filtration. Our findings provide physical insights into the rational design of functional membranes for energy and environmental applications and offer theoretical guidance and technical support for the practical application of GO membranes in separation and filtration.