This paper describes a general approach for transferring clean single-layer graphene onto silicon nitride nanopore devices and the use of the electron beam of a transmission electron microscope (TEM) to drill size-controlled nanopores in freely suspended graphene. Besides nanopore drilling, we also used the TEM to heal and completely close the unwanted secondary holes formed by electron beam damage during the drilling process. We demonstrate electron beam assisted shrinking of irregularly shaped 40–60 nm pores down to 2 nm, exhibiting an exquisite control of graphene nanopore diameter. Our fabrication workflow also rendered graphene nanopores hydrophilic, allowing easy wetting and use of the pores for studying protein translocation and protein–protein interaction with a high signal to noise ratio.